Genetically engineered Pseudomonas strains capable of metabolizing ethylene glycol and its metabolic intermediates

This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application Nos. 62/535,074 filed on Jul. 20, 2017, and 62/650,925 filed on Mar. 30, 2018, the contents of which are hereby incorporated by reference in their entirety.

The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory. The United States Government has rights in this invention pursuant to contract no. DE-AC05-00OR22725 between the United States Department of Energy and UT-Battelle, LLC.

This application contains a Sequence Listing submitted as an electronic text file entitled “NREL 17-26_ST25.txt” having a size in bytes of 84 kb and created on 18 Feb. 2021. Pursuant to 37 CFR § 1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.

During the pendency of this application, access to the invention will be afforded to the Commission upon request. Upon granting of the patent the strain will be available to the public under the conditions specified in 37 CFR 1.808. The deposit will be maintained in a public repository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer. The deposit will be replaced if it should ever become unavailable.

Polyethylene terephthalate (PET) based plastics are widely used as containers for liquids and foods and in fibers for clothing. PET plastics are largely non-biodegradable. Breakdown products of PET plastic, occurring from recycling and other degradation processes, include ethylene glycol and terephthalic acid. These breakdown products are often not fully recovered and reused during the recycling process and end up as waste.

Pseudomonas putida KT2440 demonstrates broad substrate specificity for compounds of interest to bioremediation and has high toxicity tolerance. P. putida KT2440 is often used for industrial biotechnological applications. Naturally occurring strains of P. putida KT2440 are not able to efficiently metabolize ethylene glycol or use it as a sole carbon source.

Disclosed herein are genetically engineered Pseudomonas strains capable of using ethylene glycol as a sole carbon source. In an embodiment, these Pseudomonas strains are capable of using ethylene glycol for the metabolism of compounds of interest.

In an aspect, disclosed is a genetically engineered Pseudomonas capable of growth on ethylene glycol as a sole carbon source. In an embodiment, the genetically engineered Pseudomonas has exogenous genes gcl, hyi, glxR, PP_4300, pykF, and glcDEF. In an embodiment, the genetically engineered Pseudomonas is capable of expressing an exogenous gcl (glyoxylate carboligase) operon. In another embodiment, the genetically engineered Pseudomonas is capable of expressing an exogenous glycolate oxidase. In an embodiment, the genetically engineered Pseudomonas is capable of expressing an exogenous gcl operon and expressing an exogenous glycolate oxidase operon (glcDEF). In yet another embodiment, the genetically engineered Pseudomonas is capable of expressing exogenous genes selected from the group consisting of gcl, hyi, glxR, PP_4300 and pykF. In an embodiment, the genetically engineered Pseudomonas is capable of expressing exogenous genes selected from the group consisting of gcl, hyi, glxR, PP_4300, pykF, and glcDEF. In an embodiment, the genetically engineered Pseudomonas is selected from strains MFL185, and MFL168. In an embodiment, the genetically engineered Pseudomonas is capable of growth in media containing up to about 2 M ethylene glycol. In another embodiment, the genetically engineered Pseudomonas has exogenous genes that are inserted into the genome of the Pseudomonas . In an embodiment, the genetically engineered Pseudomonas has exogenous genes that are inserted into the genome of the Pseudomonas between hpyA and PP_4218. In another embodiment, the genetically engineered Pseudomonas has exogenous genes that are under the control of an exogenous promoter. In an embodiment, the genetically engineered Pseudomonas is capable of consuming up to 0.16 g/L/h of ethylene glycol. In another embodiment, the genetically engineered Pseudomonas is capable of consuming 500 mM ethylene glycol within 120 hours. In an embodiment, the genetically engineered Pseudomonas has exogenous copies of gcl, hyi, glxR, PP_4300, and pykF, having greater than 90% identity with gcl, hyi, glxR, PP_4300, and pykF in SEQ ID NO: 4. In another embodiment, the genetically engineered Pseudomonas has exogenous copies of glcDEF having greater than 90% sequence identity with glcDEF from SEQ ID NO: 1. In an embodiment, the genetically engineered Pseudomonas has exogenous copies of gcl, hyi, glxR, PP_4300, and pykF having greater than 90% identity with gcl, hyi, glxR, PP_4300, and pykF in SEQ ID NO: 4, and has glcDEF genes having greater than 90% sequence identity with glcDEF from SEQ ID NO: 1.

In an aspect, disclosed is a genetically engineered Pseudomonas capable of growth on ethylene glycol as a sole carbon source wherein the Pseudomonas comprises exogenous genes gcl, hyi, glxR, PP_4300, pykF, and glcDEF and is capable of making polyhydroxyalkanoates. In an embodiment, the genetically engineered Pseudomonas is capable of producing polyhydroxyalkanoates at up to 0.06 grams per gram of dried cellular weight (DCW). In another embodiment, the genetically engineered Pseudomonas produces polyhydroxyalkanoates that are derived from the metabolism of ethylene glycol.

Other objects, advantages, and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.

FIG. 1 depicts a schematic of ethylene glycol metabolism and strain modifications implemented in this study in P. putida KT2440. Identified key enzymes in the metabolism of ethylene glycol are shown; a question mark represents an unidentified enzyme or putative chemical reaction responsible for conversion of a particular metabolite. Green arrows indicate the proposed ethylene glycol metabolic route of the engineered strain; the corresponding overexpressed enzymes are denoted in blue. Alternative shuttling of glyoxylate into the glyoxylate shunt is shown in grey arrows. Steps in which redox equivalents are generated are indicated in red.

FIG. 2 depicts growth and ethylene glycol utilization of engineered strains. FIG. 2A depicts growth and FIG. 2B depicts ethylene glycol utilization of plasmid bearing strains: KT2440 (pBTL2), MFL113 (pBTL2-gcl), MFL114 (pBTL2-gcl-operon), MFL115 (pBTL2-glcB), MFL116 (pBTL2-gcl-glcB) and MFL117 (pBTL2-gcl-operon-glcB). Results are given as the average of n=2 with the corresponding SEM.

FIG. 3 depicts a drawing of the overexpression constructs used: MFL168, MFL170, and MFL188 harbor the overexpression constructs integrated in the intergenic region between fpvA and PP_4218 with the constitutive tac promoter driving gene transcription. MFL168 and MFL185 contain all native genes of the gcl cluster (P tac ::gcl-hyi-glxR-ttuD-pykF), whereas MFL170 (P tac ::gcl-hyi-glxR) and MFL188 (P tac ::gcl-glxR) expresses only three or two genes of the cluster. MFL185 and MFL186 both have the tac promoter inserted before the native glycolate oxidase operon (glcDEF) and differs in that MFL185 additionally harbors the same overexpression five genes as MFL168.

FIG. 4 depicts a qRT-PCR relative gene expression compared to wild type P. putida KT2440 (2 −ΔΔCt ): Expression of gene targets from the gcl cluster (gcl, hyi, glxR, ttuD and pykF) are shown on log scale. Results are given as the average of n=2 with the corresponding SEM. Bars labeled with different letters indicate statistical significance of expression of a particular gene among the different strains (p<0.05; one-way ANOVA followed by Tukey's post hoc honest significance difference test).

FIG. 5 depicts NAD(P)H-dependent hydroxypyruvate reduction activity of whole-cell lysates derived from engineered strains using NADH-dependent and NADPH-dependent hydroxypyruvate. Results are given as the average of n=3 with the corresponding SEM. Bars labeled with different letters indicate statistical significance (p<0.05; one-way ANOVA followed by Tukey's post hoc honest significance difference test). One unit (U/mg) is defined as the amount of enzyme required to convert 1 μmol of NAD(P)H to NAD(P) + per minute.

FIG. 6 depicts growth and metabolite concentrations of the corresponding engineered strains. FIGS. 6A-6E depict the growth of and resulting concentrations of various metabolites in solutions starting with 20 mM ethylene glycol over 96 hours. FIG. 6A depicts growth (DCW g/L), FIG. 6B depicts the concentration of ethylene glycol, FIG. 6C depicts the concentration of glycolate, FIG. 6D depicts the concentration of glycolaldehyde and FIG. 6E depicts the concentration of glyoxal. FIGS. 6F-6J depict the growth of and resulting concentrations of various metabolites in solutions starting with 50 mM ethylene glycol over 96 hours. FIG. 6F depicts growth (DCW g/L), FIG. 6G depicts the concentration of ethylene glycol, FIG. 6H depicts the concentration of glycolate, FIG. 6I depicts the concentration of glycolaldehyde and FIG. 6J depicts the concentration of glyoxal. Results are given as the average of n=2 with the corresponding SEM.

FIG. 7 depicts growth toxicity studies of P. putida KT2440 in the presence of 20 mM glucose in M9 minimal medium supplemented with various concentrations of metabolites. FIG. 7A depicts the growth of P. putida KT2440 in the presence of 0, 25, 50, and 100 mM ethylene glycol, FIG. 7B depicts the growth of P. putida KT2440 in the presence of 0, 12.5, 25 and 50 mM sodium oxalate, FIG. 7C depicts the growth of P. putida KT2440 in the presence of 0, 2, 4, and 8 mM glycolaldehyde, FIG. 7D depicts the growth of P. putida KT2440 in the presence of 0, 25, 50, and 100 mM sodium glycolate, FIG. 7E depicts the growth of P. putida KT2440 in the presence of 0, 25, 50, and 100 mM sodium glyoxylate, and FIG. 7F depicts the growth of P. putida KT2440 in the presence of 1, 2, 3, and 5 mM glyoxal. Results are given as the average of n=3 with the corresponding SEM.

FIG. 8 depicts the growth of P. putida KT2440, MFL168, MFL186 and MFL185 and various metabolite concentrations in the media starting with 50 mM ethylene glycol over 96 hours. FIG. 8A depicts growth in DCW g/L, FIG. 8B depicts the concentration of ethylene glycol, FIG. 8C depicts the concentration of glycolate, FIG. 8D depicts the concentration of glycolaldehyde, and FIG. 8E depicts the concentration of glyoxal. Results are given as the average of n=2 with the corresponding SEM.

FIG. 9 depicts growth of engineered strains under various conditions. FIG. 9A depicts growth of MFL168 in M9 minimal medium containing 0, 20, 40, 60, and 80 mM concentrations of ethylene glycol. FIG. 9B depicts growth of MFL185 in M9 minimal medium containing concentrations of ethylene glycol of 0.5, 1.0, 1.5, 1.8, and 2 M as a sole carbon source as measured in the Bioscreen C. FIG. 9C depicts growth of MFL185 in M9 minimal medium containing concentrations of 0.5 M and 1 M ethylene glycol in shake flasks measuring DCW (g/L) over time. FIG. 9D depicts ethylene glycol utilization over time of MFL185 in M9 minimal medium containing concentrations of 0.5 M and 1 M ethylene glycol in shake flasks. In FIGS. 9C and 9D , data from cultures grown in 1 M ethylene glycol with twice M9 salts and a higher cell inoculum are designated by dashed lines. Results are given as the average of n=2 with the corresponding SEM.

FIG. 10 depicts images of engineered strains, as well as quantity and type of mcl-PHAs produced. FIG. 10A depicts bright field (BF) microscopy of MFL185 cells after growth in ethylene glycol and fluorescent microscopic observation of mcl-PHAs via Nile Red (NR) staining of same cells. FIG. 10B depicts mcl-PHAs production on a dry cell weight basis. FIG. 10C depicts the composition of various chain length mcl-PHAs produced from ethylene glycol and acetate. Results are given as the average of n=3 with the corresponding SEM.

FIG. 11 depicts a comparison of mcl-PHAs production by MFL185 grown in 100 mM acetate or ethylene glycol. FIG. 11A depicts curves of strains over time. FIG. 11B depicts time-course of mean fluorescence of Nile Red stained cells. FIG. 11C depicts fluorescent microscopic observation of Nile Red stained MFL185 cells after 72 hours cultured in ethylene glycol. Results are presented as means±SEM (n=3).

FIG. 12 depicts a prediction of transcriptional units (Database of PrOkaryotic OpeRons (DOOR)) is depicted here for genes in the proximity of the gcl operon. Predicted transcripts are indicated by grey arrows. In FIG. 12 , the prediction or known function of the corresponding ORF are indicated below the predicted transcripts. The presence of two inverted repeats are shown as IR-1 (SEQ ID NO: 49) or IR-2 (SEQ ID NO: 50).

FIG. 13 depicts synergistic inhibitory effect of glycolaldehyde and glyoxal. Growth assays were conducted in 100 well plates in the Bioscreen C MBR analyzer. Individual wells of the plate were filled with 200 μL of M9 medium-containing 20 mM glucose and the respective concentration of glycolaldehyde and glyoxal according to a 3-level full-factorial design (Table 3), growth rates expressed as means±SEM (n=3). Bars labeled with different letters indicate statistical significance of different runs (p<0.05; one-way ANOVA followed by Tukey's post hoc honest significance difference test).

FIG. 14 depicts co-feeding of P. putida KT2440 in C-limited chemostat cultivations on mineral medium with 30 mM acetate supplemented with 30 mM ethylene glycol (black), 30 mM glyoxylate (green), or no co-feed (grey). FIG. 14A depicts a comparison of biomass (OD 600 ) at steady state. FIG. 14B depicts extracellular metabolites at steady state. The metabolites profile of the acetate control is not shown since none were detected. FIG. 14C depicts flux analysis of redox equivalent-yielding metabolism of ethylene glycol (upper value) or glyoxylate (lower value) in mM gCDW −1 h −1 . Cells were grown at a dilution rate of 0.2. Error bars indicate the deviation from the mean (n=2).

FIG. 15 depicts adaptive laboratory evolution of P. putida KT2440 on ethylene glycol and characterization of adapted strains. FIG. 15A depicts sequential batch cultivation on mineral medium with 15 mM ethylene glycol. Arrows indicate the time points where strains were isolated. FIG. 15B depicts extracellular metabolic products and FIG. 15C depicts biomass growth of the isolated ALE strains E1.1 and E6.1 growing on 30 mM ethylene glycol. FIG. 15C also depicts of P. putida KT2440 and all adapted strains in a shake flask cultivation on minimal medium with 30 mM ethylene glycol. FIG. 15D depicts growth comparison of P. putida KT2440 and all adapted strains in minimal medium containing 30 mM ethylene glycol (in light colors) and 120 mM ethylene glycol (in darker colors). Growth was detected via a growth profiler in 96-square-well plates. Error bars indicate the deviation from the mean (n=2).

FIG. 16 depicts genomic and transcriptomic analysis of ethylene glycol adaptive mutations in P. putida KT2440. FIG. 16A depicts genomic context of mutations involved in the activation of the gcl pathway (coordinates: 4866804-4902814, 36 kb), and FIG. 16B depicts a beta-oxidation operon (coordinates: 2325342-2334253, 9 kb). Mutations and their effects are indicted below the affected site. Black arrows indicate transcriptional regulators, grey arrow indicate putative transporters, ochre arrows indicate functions related to beta-oxidation, and white arrows indicate miscellaneous unrelated functions. Other arrow colors correspond to metabolic functions shown in FIG. 16C depict a simplified representation of purine metabolism via allantoin and glyoxylate. FIG. 16D depicts a box-and-whisker plot of relative expression levels of genes implicated in ethylene glycol metabolism in P. putida KT2440 wt and evolved mutants determined by qRT-PCR. The 2ΔCt values were normalized to rpoD. Individual data points are plotted onto the graph, whiskers indicate minimum to maximum values.

FIG. 17 depicts growth of P. putida KT2440 and ALE strains E6.1 and E6.2 after 25 h in mineral medium containing 20 mM allantoin and/or 20 mM ethylene glycol (EG). The label ‘—NH 4 + ’ indicates that ammonium was omitted from the medium, leaving allantoin as sole nitrogen source. Error bars indicate the deviation from the mean (n=2).

FIG. 18 depicts comparative growth of strains. FIG. 18A depicts a comparison of strains made by reverse engineering over a 30 minute period of time. FIG. 18B depicts a comparison of strains made by reverse engineering over a 60 minute period of time.

FIG. 19 depicts performance of integrated strains in M9 minimal medium containing 50 mM ethylene glycol. FIG. 19A depicts growth of integrated strains in M9 minimal medium containing 50 mM ethylene glycol in DCW (g/L). FIG. 19B depicts ethylene glycol utilization. FIG. 19C depicts the concentration of glycolate. FIG. 19D depicts the concentration of glycolaldehyde. FIG. 19E depicts the concentration of glyoxal.

FIG. 20 depicts toxicity assays using the Bioscreen C with P. putida KT2440 performed in minimal medium containing 20 mM glucose and the following substrates or metabolites: FIG. 20A depicts growth with 0, 25, 50 and 100 mM ethylene glycol; FIG. 20B depicts growth with 0, 12.5, 25, and 50 mM sodium oxalate; FIG. 20C depicts growth with 0, 2, 4 and 8 mM of glycolaldehyde; FIG. 20D depicts growth with 0, 25, 50 and 100 mM glycolate; FIG. 20E depicts growth with 0, 25, 50 and 100 mM sodium glyoxylate; and 20 F depicts growth with 1, 2, 3, and 5 mM glyoxal.

FIG. 21 depicts growth and metabolite concentrations of the corresponding overexpression strains with 20 mM ethylene glycol. FIG. 21A depicts growth in DCW (g/L). FIG. 21B depicts ethylene glycol concentration. FIG. 21C depicts glycolate concentration. FIG. 12D depicts glycolaldehyde concentration.

FIG. 22 depicts characterization of a glycolate oxidase overexpression strain that is also overexpressing the gcl operon. FIG. 22A depicts growth of P. putida KT2440, MFL168, MFL186 and MFL185 in 50 mM ethylene glycol. FIG. 22B depicts the concentration of ethylene glycol. FIG. 22C depicts the concentration of glycolate. FIG. 22D depicts the concentration of glycoaldehyde.

FIG. 23 depicts growth of engineered strains under various growth conditions. FIG. 23A depicts the growth of MFL168 in M9 minimal medium containing varying concentrations of ethylene glycol only as measured in the Bioscreen C. FIG. 23B depicts the growth of MFL 185 in M9 minimal medium containing varying concentrations of ethylene glycol only as measured in the Bioscreen C. FIG. 23C depicts growth of MFL185 in M9 minimal medium containing varying concentrations of ethylene glycol shake flasks measuring DCW (g/L) over time. FIG. 23D depicts ethylene glycol utilization over time.

FIG. 24 depicts fold change in gene expression of various gene targets by qRT-RT experiments compared to the control P. putida KT2440. Expression of gene targets from the gcl operon (gcl, hyi, glxR, PP_4300 and pykF) in addition to the native hydroxypyruvate reductase (PP_0762). Expression levels were normalized to the housekeeping gene, rpoD and then normalized to expression levels present in the wild-type strain P. putida KT2440.

FIG. 25 depicts NAD(P)H-dependent hydroxypyruvate reduction activity of whole lysate protein of engineered strains using NADH-dependent and NADPH-dependent hydroxypyruvate reduction activity of whole cell lysates. Results are the average of n=3, bars labelled with different letters indicate statistical significance (p<0.05; one-way ANOVA followed by Tukey's post hoc honest significance difference test). One unit (U/mg) was defined as the amount of enzyme required to convert 1 μmol of NAD(P)H to NAD(P)+ per minute.

Wildtype P. putida KT2440 cannot grow on ethylene glycol as a sole carbon source. As disclosed herein, adaptive laboratory evolution experiments performed on wildtype strains of P. putida resulted in non-naturally occurring P. putida KT2440 mutants that can use ethylene glycol as their sole carbon source. The metabolism of ethylene glycol and its derivatives plays a pivotal role in the biotechnological utilization of plastic waste and lignin, and its oxidation products glycolic and glyoxylic acid are valued commodity chemicals. The characterization of the metabolic pathways in these mutants is useful for engineering Pseudomonas strains for the production of compounds of interest, and for using ethylene glycol as a redox energy yielding co-substrate. In an embodiment, the genetically engineered Pseudomonas strains disclosed herein can be used for the metabolism of glycoaldehyde.

A metabolic engineering approach to convert ethylene glycol into cellular biomass is disclosed herein. Although, P. putida KT2440 has all the genes necessary for growth in ethylene glycol, it is prevented from doing so, by its own regulation. By overexpressing the gcl operon, ethylene glycol can be used for cell growth. An exogenous gene or genes have been introduced into a genetically engineered organism and/or integrated into the genome of a genetically engineered organism resulting in a non-naturally occurring, genetically engineered organism. In an embodiment the engineered organisms disclosed herein express and/or overexpress glc operon and/or gcl operons that are exogenous.

In an embodiment, the genes introduced into the genetically engineered Putida organisms disclosed herein are exogenous. The exogenous genes may be additional copies of native genes in the genetically engineered organism. In an embodiment the genes introduced into engineered Putida strains disclosed herein are additional copies of native genes. In an embodiment, the genes introduced into engineered Putida strains disclosed herein are additional copies of native genes and are under the control of exogenous promoters.

As depicted herein, the quantitative physiological characterization of ethylene glycol co-metabolism by P. putida KT2440 provides valuable insights for the production of the latter value-added chemicals, and also identifies opportunities and bottlenecks for the use of ethylene glycol as a redox energy yielding co-substrate.

Experiments were performed to expand the ability of P. putida to both use and produce substrates of industrial interest by enhancing its metabolism of ethylene glycol via metabolic engineering through the overexpression of existing regulated pathways that include the glyoxylate carboligase (gcl) operon. Growth in concentrations of ethylene glycol above 50 mM was inhibited by the accumulation of toxic intermediates, glycolaldehyde and glyoxal. The additional overexpression of glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting the growth in up to 2 M (120 g/L) and consumption of greater than about 0.5 M (30 g/L) of ethylene glycol.

Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida , we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (˜124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, the methods and non-naturally occurring genetically modified organisms disclosed herein result in P. putida KT2440 strains that are now useful for ethylene glycol consumption, and can serve as a biocatalyst for applications in the remediation of waste polyester plastics and biomass-derived wastewater streams.

Ethylene glycol is a large-volume industrial chemical used for myriad applications including for the production of polyester plastics such as polyethylene terephthalate (PET), as a coolant in antifreeze, as a deicing fluid for aircraft, and as an inhibitor of clathrate hydrate formation in natural gas pipelines. Because of its widespread use, it is a common pollutant in the environment, where it is broken down either chemically or biologically. Plastic wastes, including ethylene glycol and terephthalic acid, offer novel substrates for industrial biotechnology to convert into value-added products, especially given the worldwide concern over plastic accumulation in the biosphere.

Aerobic ethylene glycol metabolism also generates the highly toxic intermediate glycolaldehyde. Besides being a metabolic intermediate in ethylene glycol metabolism, this compound is also often a significant component of lignocellulose-derived streams, and can also be present in pyrolysis wastewater in concentrations as high as 50 g/kg. Currently, wastewater streams containing significant amounts of glycolaldehyde from biomass pyrolysis are sent to thermal wastewater treatment processes, but converting this carbon to a value-added co-product stream would improve biorefinery economics.

From an environmental perspective, disposal of ethylene glycol or its intermediate, glycolaldehyde, poses serious environmental problems. Soil bacteria, such as pseudomonads, are likely responsible for a substantial extent of ethylene glycol catabolism in the environment. The obligate aerobic organism P. putida KT2440 uses ethylene glycol only as a source for the production of reducing equivalents and energy, whereas ethylene glycol enables biomass formation in other P. putida strains, such as JM37. Through comparative proteomics of these two P. putida strains, the catabolism of ethylene glycol in P. putida KT2440 was proposed to proceed via two functionally redundant, periplasmic quinoproteins PedE and PedH and the subsequent activity of the two cytoplasmic aldehyde dehydrogenases PP_0545 and PedI, together with the membrane anchored oxidase GlcDEF, yielding glyoxylic acid ( FIG. 1 ). Glyoxylic acid was then proposed to be further metabolized either through the dicarboxylic acid pathway initiated by ligation to acetyl-CoA catalyzed by malate synthase (GlcB) or through the partial use of TCA-cycle reactions initiated by the AceA-dependent ligation of glyoxylate and succinate to yield isocitrate (glyoxylate cycle). However, the metabolic regeneration of either of these adducts yields two molecules of CO 2 , and thus the metabolism of glyoxylate through this cycle will not enable growth. In contrast, the catabolic pathway that allows biomass formation in strain JM37 proceeds through the glyoxylate carboligase, (Gcl) pathway, and energy generation through the glyoxylate shunt through malate synthase (GlcB) and isocitrate lyase (AceA).

In contrast to the productive use of ethylene glycol by other strains of P. putida , the lack of growth of P. putida KT2440 on ethylene glycol is puzzling, as the organism exhibits the genomic inventory to use ethylene glycol as a carbon source through the initial ligation of two glyoxylate molecules to tartronate semialdehyde by the glyoxylate carboligase (Gcl) enzyme. According to the Pseudomonas Genome Database predictions, the enzymes adjacent to gcl encode hydroxypyruvate isomerase (hyi), tartronate semialdehyde reductase (glxR), hydroxypyruvate reductase (ttuD), and pyruvate kinase (pykF), which could, together with glycerate kinase (garK) encoded on a different locus of the genome, allow conversion of glyoxylic acid into biomass. The lack of growth with ethylene glycol in native, naturally occurring P. putida KT2440 is caused by an unknown regulatory mechanism, which prevents the functional production of the aforementioned enzymes necessary to use ethylene glycol for growth. As disclosed herein, by using adaptive laboratory evolution techniques, it was discovered that the observed lack of growth with ethylene glycol is caused by an unknown regulatory mechanism wherein repression of gcl-operon genes is overcome by a mutation in a specific transcriptional regulator.

In the present disclosure, we demonstrate that efficient glyoxylate and ethylene glycol conversion into biomass involves the constitutive expression of gcl in addition to genes in proximity of gcl. Contrary to operon prediction software, transcriptomic analysis reveals that the operon consists of four other genes contiguous to gcl (hyi, glxR, ttuD, and pykF), permitting a different and more effective path from glyoxylate to glycerate, via hydroxypyruvate. An additional bottleneck was discovered at the metabolite glycolate as concentrations of substrate increased, resulting in the accumulation of a toxic intermediate (glycolaldehyde). The overproduction of the native glycolate oxidase operon (glcDEF) resolves this bottleneck, which leads to increased metabolic flux, and decreases the accumulation of toxic intermediates, transforming P. putida KT2440 into an efficient ethylene glycol-metabolizing strain. Also, disclosed herein are engineered strains that enable efficient conversion of ethylene glycol into medium-chain-length polyhydroxyalkanoates (mcl-PHAs), a high value chemical building block. Engineered P. putida strains disclosed herein can serve as a foundation for conversion of both ethylene glycol from plastic waste and glycolaldehyde in biomass-derived wastewater streams.

In an embodiment, disclosed herein are engineered P. putida strains that are capable of the expression of two exogenous genes (gcl and glxR) that confer the ability of engineered P. putida to grown on ethylene glycol. In an embodiment, engineered P. putida strains are disclosed that comprise a gcl operon that contains the genes gcl, hyi, glxR, ttuD, and pykF that allows for growth and/or improved growth in ethylene glycol. In another embodiment, P. putida strains are disclosed herein that are capable of overexpression of glycolate oxidase which removes a glycolate metabolic bottleneck. In an embodiment, disclosed herein are engineered P. putida KT2440 strains that consume up to about 31 g/L of ethylene glycol. In another embodiment, disclosed herein are engineered P. putida KT2440 strains capable of producing mcl-PHAs from ethylene glycol.

Plasmid Construction

Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers synthesized by Integrated DNA Technologies (IDT) were used in all PCR amplification. Plasmids were constructed using Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer's instructions. Primers used for PCR amplification and Gibson assembly are listed in Table 1.

Table 1 contains primers used for creating plasmid and integration constructs. Underlined nucleotides denote homology to a target gene. Nucleotides in bold font are restriction sites.

The plasmid, pBTL2 was used as the backbone of all plasmid-based overexpression constructs in engineered P. putida KT2440 strains whose dried cellular weight (DCW) and use of ethylene glycol is depicted in FIG. 2 . In an embodiment, features of the plasmids used include the lac promoter and a soxR terminator. Plasmids were constructed by amplifying the plasmid (pBTL2) or gene(s) of interest from P. putida KT2440, digesting with appropriate restriction enzymes then ligating vector and inserts to produce plasmids pBTL2-gcl, pBTL2-gcl-operon, pBTL2-glcB, pBTL2-gcl-glcB, pBTL2-gcl-operon-glcB (pMFL113-117, respectively); strain name designations are listed in Table 2.

Plasmids for gene integration were constructed in plasmid pK18mobsacB from ATCC (American Type Culture Collection, Manassas, Va.), which is unable to replicate in P. putida KT2440, and contains the kanamycin-resistant marker to select for integration of the plasmid into the genome by homologous recombination and sacB to counterselect for a second recombination event to subsequently remove the plasmid backbone from the genome.

The plasmids, pMFL160 and pMFL161, used for of integration of operons containing the gene gcl in strains MFL168, MFL170, MFL185, and MFL188 were constructed based on the integration vector pK18mobsacB. They contain the 1 kb homology region on either side of the intergenic region immediately after the fpvA (outer membrane ferripyoverdine receptor) terminator and PP_4218 (lipase/esterase) of P. putida KT2440 (see Table 1 for primers used for construction).

Features include the tac promoter to drive gene expression and a tonB terminator situated behind the fragments cloned into the plasmid backbone, which are depicted in FIG. 3 . The ribosomal binding site (RBS) (GAGGAGGA) in front of gcl was predicted to have a translation initiation rate (TIR) of 2700 using an online RBS calculator and was the same for MFL168, MFL170, and MFL185. We used the optimal RBS (AAGGAGGT) for expression of gcl and glxR in MFL188. The sequences of all other promoter regions, genes, or operons remained unchanged, and thus represent the native chromosomal sequence. The overexpression of glycolate oxidase genes (glcDEF) in plasmid pLJ030, which was used to construct strains MFL185 and MFL186, was achieved by integrating the tac promoter upstream of the glycolate oxidase operon (glcDEFG_PP_3749) and behind the native promoter, while additionally optimizing the RBS for glcD (AAGGAGGT). The nucleotide plasmid sequences are SEQ ID NO: 1 for pLJ030, SEQ ID NO: 2 for pLJ032, SEQ ID NO: 3 for pMFL160, and SEQ ID NO: 4 for pMFL161. Plasmids were transformed into competent NEB 5-alpha F′P E. coli (New England Biolabs) according to the manufacturer's instructions. Transformants were selected on LB plates containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 15 g/L agar, supplemented with 50 μg/mL kanamycin grown at 37° C. The sequences of all plasmid inserts were confirmed using Sanger sequencing.

Strain Construction

P. putida KT2440 (ATCC 47054) was used as the strain for engineering and gene replacements that were made using the antibiotic/sacB system of selection and counter-selection. To prepare electrocompetent cells of different P. putida KT2440 strains, we used a modified protocol. Briefly, cultures were grown overnight in LB broth and incubated at 30° C., shaking at 225 rpm. The next day, cells were centrifuged 21,130×g in an Eppendorf centrifuge for 1 minute at room temperature, washed three times in 0.3 M sucrose in half the original volume. Finally, the cells were resuspended in 1/50 th of the culture's original volume in 0.3 M sucrose. Cells were immediately used for electroporation by introducing 5 μL (200 ng-2 μg) of plasmid DNA to 50 μL of the electrocompetent cells, transferred to a chilled 0.1 cm electroporation cuvette, and electroporated at 1.6 kV, 25 μF, 200Ω. Subsequently, 950 μL SOC (NEB) was added and the cells were incubated shaking at 225 rpm, 30° C., for 2 h. The entire transformation was plated on an LB agar plate containing appropriate antibiotics and incubated at 30° C. overnight. Initial colonies from the transformation plates were re-streaked on selective LB agar plates and grown at 30° C. overnight to obtain clonal transformants. For sucrose counter-selection, clonal transformants were streaked on YT plates containing 25% (YT+25%; w/v) sucrose (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, 18 g/L agar), and incubated at 30° C. overnight. P. putida KT2440 containing the sacB gene can grow on YT+25% sucrose media. Therefore, single colonies presumed to have lost the sacB gene via homologous recombination, indicated by larger colonies, were picked and re-streaked on fresh YT+25% sucrose plates and incubated at 30° C. overnight to finally obtain clonal sucrose resistant and antibiotic sensitive strains. All strains were analyzed for the correct gene replacement by performing a colony PCR at the site of integration. Table 2 lists the specific strains produced in this work and the plasmids used for the integration.

Culture Growth and Metabolite Analysis

Shake flask experiments were performed using M9 minimal media (Sigma-Aldrich) containing 6.78 g/L disodium phosphate, 3 g/L monopotassium phosphate, 0.5 g/L NaCl, 1 g/L NH 4 Cl, 2 mM MgSO 4 , 100 μM CaCl 2 ), and 40 μM FeSO 4 .7H 2 O supplemented with 20 mM glucose (Fisher Scientific), ethylene glycol, or sodium acetate (Sigma-Aldrich). For analysis of mcl-PHA production, nitrogen-limiting M9 medium was prepared by substituting 1 g/L of NH 4 Cl with 0.132 g/L of (NH 4 ) 2 SO 4 (Sigma-Aldrich). For growth experiments with ethylene glycol, glyoxylate, or glycolaldehyde, overnight cultures were harvested, washed in M9 minimal media without a carbon source, and used for inoculation of fresh medium to an OD 600 of 0.1 and at OD 600 of 0.5 for cultures grown in 2×M9 salts. Cultures were grown with a volume of 25 mL in 125 mL baffled shake flasks, incubated at 30° C. with shaking at 225 rpm. Growth of the cultures was followed by periodic measurement of the optical density at 600 nm (OD 600 ) using a Beckman DU640 spectrophotometer (Beckman Coulter, Brea Calif.). The dry cell weight of samples (DCW) was calculated by using the conversion factor y=0.5746x, where y is DCW in g/L and x=OD 600 , supported by experimental data that included OD 600 measurement values <3.3. Except for experiments conducted with plasmid-bearing strains, all shake flask cultures were performed in duplicate. Concentrations of glucose, ethylene glycol, glycolaldehyde, glyoxal, glycolate, glyoxylate, and oxalate in sterile-filtered culture supernatants were measured with high performance liquid chromatography (HPLC) on an Agilent1100 series system (Agilent USA, Santa Clara, Calif.) utilizing a Phenomenex Rezex RFQ-Fast Fruit H+ column (Phenomenex, Torrance, Calif.) and cation H+ guard cartridge (Bio-Rad Laboratories, Hercules, Calif.) at 85° C. A mobile phase of 0.1 N sulfuric acid was used at a flow rate of 1.0 mL/min and a diode array detector was utilized for compound detection. Products were identified by comparing the retention times and spectral profiles with pure compounds and were calculated based on a calibration curve generated for each compound. To quantify yield and composition of mcl-PHAs as a percent of the dry cell weight in cultures growth in media containing ethylene glycol or acetate, shake-flask experiments were performed in 250-mL Erlenmeyer flask filled with 50 mL of nitrogen-limiting M9 medium-containing 100 mM of ethylene glycol. The detailed descriptions of microscopic observations and analytical quantification of mcl-PHA are described below.

mcl-PHA Observation, Quantification, and Characterization

Medium chain length PHA formation in P. putida KT2440 was observed by using an epifluorescence Nikon Eclipse 80i microscope. One mL samples were taken from ethylene glycol-containing shake flask cultures. Cells were pelleted by centrifugation at 13,000 rpm for 1 min, washed twice with 1× phosphate buffer saline (PBS), resuspended in 1 mL PBS-containing 10 μg/mL Nile Red (Molecular probes, Invitrogen Cooperation, USA), and incubated at room temperature in the dark for 30 min. Cells were pelleted again, washed twice with 1×PBS, and resuspended in 1 mL PBS. 5 μL of resuspended cells were mixed with 5 μL of 1% (w/v) low-melting-temperature agarose to immobilize the cells and placed on a microscopic slide with coverslip. Nile Red fluorescence was observed with band-pass filtering between 560-590 nm.

Fluorescence-activated cell sorting (FACS) analysis of mcl-PHAs: Time course of mcl-PHA production of P. putida was monitored using a BD FACSAria (BD Biosciences, USA) instrument equipped with BD FACSDiva data acquisition and analytical software. After cells were stained with Nile Red as previously described, cell pellets were washed twice with PBS solution and resuspended in BD FACSFlow sheath fluid (BD Biosciences, USA) for analysis. Samples were loaded into FACS, mcl-PHA content of cells were monitored using the 488 nm (exited) laser coupled with B610-20A (610 nm) detection channels, and 20,000 events were recorded to generate the histograms for obtaining mean fluorescents.

Analysis of mcl-PHA quantity and composition: Extraction and quantification of mcl-PHAs in P. putida was performed. Samples were prepared for derivatization by adding about 30 mg of biomass to a gas chromatography glass (GC) vial. To track derivatization, 25 μL of benzoic acid (Sigma Aldrich) dissolved in dichloromethane (8 mg/mL) was added as an internal surrogate. Samples were derivatized by adding about 1 mL of 20% BF 3 /MeOH to the GC vial, which was sealed, vortexed, and placed in a heating block at 80° C. for about 16 hrs. Vials were then removed from the heating block and allowed to cool to room temperature. Vial contents were pipetted into a 10 mL volumetric flask and the vial residual was rinsed twice with dichloromethane (DCM) (Sigma Aldrich) before filling the flask to 10 mL total with additional DCM. The 10 mL solution was transferred to a (polytetrafluoroethylene) PTFE capped vial and about 3 mL of water was added to form a bi-phase and vortexed to wash out residual BF 3 to the aqueous layer. The DCM layer (2 mL) was then transferred into another gram vial containing Na 2 SO 4 and Na 2 CO 3 to dry and neutralize any remaining BF 3 . The dried and neutralized solutions were syringe filtered (0.2 μm PTFE) into fresh GC vials for analysis. To track recovery of PHAs during sample derivatization and analysis, triplicate biomass samples of P. putida KT2440 grown at NREL were processed in parallel. Recovery yields during sample workup were calculated based on a cell dry weight PHA content of 24% determined by bulk sample solvent extraction. Hydroxyacid methyl esters were identified and the distribution quantified by gas chromatography mass spectroscopy (GC-MS) using an Agilent 6890N GC equipped with a 5973 MSD (Agilent Technologies). Agilent MSD Productivity Chemstation G1701 software was used to collect and quantitate analytes. 8-Hydroxyoctanoic acid methyl ester, 10-hydroxydecanoic acid methyl ester, 12-hydroxydodecanoic acid methyl ester, and 14-hydroxytetradecanoic acid methyl ester were obtained from Matreya (98+ percent purity, Matreya, State College, Pa., USA), and used to determine the GC-MS instrument response. Samples were injected at a volume of 1 μL onto a Stabilwax-DA (Restek, Bellefonte, Pa.) column (30 m×0.25-mm id, 0.25-μm film) in splitless mode, with helium at 1 mL/min constant flow used as the carrier gas. The GC/MS method consisted of a front inlet temperature of 250° C., and an auxiliary transfer line temperature of 260° C. The separation used had a starting temperature of 35° C. and this was held for 5 min, then ramped at 15° C./min to a temperature of 225° C. and held for 2.0 minutes, then ramped at 15° C./min to a temperature of 250° C. and held for 5.67 minutes with a solvent delay time of 6 minutes, for a total run time of 27 min. Sample total ion counts were collected on the mass spectrometer at scan range from 30 to 450 m/z. Calibration curves where made by using target ions and diluting the derivatized standards between a concentration of 10-1000 μg/L. A minimum of seven calibration levels was used resulting in an r 2 coefficient of 0.995 or better for each analyte and a check calibration standard (CCS) was analyzed every ten samples to insure the integrity of the initial calibration. An internal standard of 1,2-diphenylbenzene (99.9+ percent purity, AccuStandard, New Haven, Conn.) was added to all standards and samples at a concentration of 20 ug/L to adjust for any detector response changes.

Toxicity Tests and Competitive Inhibition Assays

Toxicity tests and competitive inhibition assays were performed using Bioscreen C MBR analyzers (Growth Curves US, Piscataway, N.J.). For toxicity tests, overnight cultures of P. putida KT2440 were grown in M9 medium containing 20 mM glucose starting at an OD 600 of 0.05-0.1 at 30° C. with shaking at 225 rpm in baffled shake flasks until the OD 600 reached about 1.0-1.5. Cells were subsequently concentrated by centrifugation and inoculated into wells of Bioscreen C microplates at an initial OD 600 =0.05. Each well contained a total volume of 300 M9 medium, 20 mM glucose, and inhibitors at various concentrations. Incubations were performed at 30° C. with maximum shaking. Absorbance readings were taken every 15 min. For competitive inhibition assays, individual wells of the plate were filled with 200 μL of M9 medium containing 20 mM glucose and a respective concentration of glycolaldehyde and glyoxal according to full-factorial test run results of which are presented in Table 3 and depicted in graphical form in FIG. 13 . Initial OD 600 was set at 0.1 by using an overnight culture of wild type P. putida KT2440. Samples were incubated as described above. Operation of the Bioscreen C MBR and collection of turbidity measurements (OD 420-580 ) were computer automated with EZ Experiment.

RNA Extraction, cDNA Synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

To prepare P. putida cultures for RNA extraction, cells were grown overnight in M9 minimal medium containing 20 mM glucose in baffled shake flasks at 30° C., 225 rpm. Cells were then diluted and used to inoculate fresh cultures containing 20 mM ethylene glycol and 40 mM sodium acetate to an initial OD 600 of 0.1. After incubation at 30° C. with shaking at 225 rpm to mid-exponential growth phase (OD 600 0.8-1), 2× volume of Qiagen RNAprotect Bacteria Reagent was added to the cultures and allowed to mix for 5 minutes. Subsequently, cells were harvested by centrifugation at 5,000×g for 15 min at 4° C. Supernatant was removed and cells were frozen and stored at −80° C. until further analysis. Supernatants of cultures prior to addition of RNAprotect reagent was analyzed for acetate and ethylene glycol by HPLC that showed that substrate was still available. RNA was extracted from cells using Qiagen's RNeasy mini kit following manufacturer's instructions including a DNAse (Qiagen RNase-Free DNase) in column digestion for one hour at room temperature following manufacturer's instructions. After one round of RNA isolation, a DNase digestion was performed (TURBO DNase; Ambion, Austin, Tex., USA). After two hours incubation at 37° C., the DNase was removed from the RNA sample with an additional purification step using the Qiagen's RNeasy mini kit. cDNA was prepared from the purified RNA using an iScript Reverse Transcription supermix kit for RT-qPCR (Bio-Rad). The expression levels of seven genes were analyzed using primers designed by the Realtime PCR tool for RT-qPCR and is listed in Table 4.

Quantitative RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Lab, Hercules, Calif., USA). The reaction conditions were 10 min at 95° C., 39×(15 s at 95° C., 45 s at 55° C., followed by melting curve analysis: 1 min at 95° C., 81×(30 s starting at 55° C., increasing 0.5° C. per cycle, ending at 95° C.). Experiments were performed in triplicate with biological duplicates. The gene expression levels were assessed by comparing the Ct value of the house keeping gene rpoD to the Ct value of the target gene using the following formula:

Gene expression level=2 Ct(rpoD)−Ct(target)

Ct values represent the first cycle at which the instrument can distinguish the fluorescence of nucleic acid amplification generated as being above the background signal. Final expression levels were averaged for each target gene and normalized to the expression level of the control ( P. putida KT2440) strain.

Cell Preparation, Extraction, and NAD(P)H Oxidizing Activity Assays

For extracting whole lysate protein for enzyme assays, strains were grown in LB medium and harvested by centrifugation at 3,828×g for 5 min at 4° C. during exponential growth phase. After washing with water twice, cells were finally re-suspended in B-PER (Thermo Fisher Scientific, Waltham, Mass., USA) solution supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Whole cell lysates were obtained by following the manufacturer recommended protocol (Thermo Fisher Scientific). The protein concentration of samples was assessed using a NanoDrop 2000/c Spectrophotometer (Thermo Fisher Scientific) by following the manufacturer protocol. Hydroxypyruvate reduction activity of samples was measured by monitoring oxidation of NAD(P)H at 340 nm with FLUOstar Omega micro plate reader (BMG Labtech, Ortenberg, Germany). For this, two hundred μL of a reaction mixture-containing 150 μL of 50 mM potassium phosphate buffer (pH 7), 20 μL of 0.5 M lithium (3-hydroxypyruvate hydrate (Sigma-Aldrich, St. Louis, Mo., USA), 20 μL of 0.7 mM NAD(P)H, and 10 μL of the cell extracts were used for the enzyme activity assay. One unit (1 U) of enzyme was defined by the conversion of 1 μM of NAD(P)H into NAD(P) + per minute. The units were normalized to the total protein content of the corresponding sample (mg).

Statistical Analysis

All experiments, except the initial plasmid-bearing strains, were performed in duplicate or triplicates as mentioned in figure legends. The results are expressed in mean values and standard errors of the means (SEM). A one-way analysis of variance (ANOVA) followed by Tukey's post hoc honest significance difference test was adopted for multiple comparisons. Data analysis was performed using KaleidaGraph statistical program (Synergy Software, PA, USA). For a pair-wise comparison of the differences between the sample averages of two groups, a one-tailed Student's t-test without known deviations was employed.

Growth of P. putida KT2440 Plasmid-Bearing Strains in Ethylene Glycol

Given that the gene cluster containing gcl, hyi, glxR, ttuD, and pykF together with glcB are needed for glyoxylic acid catabolism in P. putida KT2440, experiments were performed to identify the minimal enzymatic setup which would allow growth of the organism. As such, various combinations of gcl or the gcl cluster and glcB were cloned into the plasmid pBTL2 under the control of a lac promoter and transformed into P. putida KT2440. Based on the Database of Prokaryotic Operons (DOOR), the gene glcB is predicted to represent a single transcriptional unit ( FIG. 12 ). In contrast, gcl is predicted to be co-transcribed along with hyi, the gene that encodes hydroxypyruvate isomerase. In proximity and predicted to be in two additional transcripts are three additional genes, namely glxR, ttuD, and pykF, which are annotated by the Biocyc database as tartronate semialdehyde reductase, a hydroxypyruvate reductase, and a pyruvate kinase, respectively. As a consequence, Hyi, which catalyzes the isomerization between hydroxypyruvate and tartronate semialdehyde and ttuD could catalyze a reduction reaction from hydroxypyruvate to glycerate that would provide an alternative route from tartronate semialdehyde to glycerate via hydroxypyruvate, see FIG. 1 . The GlxR protein is also likely important as it enables the conversion of tartronate semialdehyde directly to glycerate.

Strains constitutively expressing gcl (MFL113), glcB (MFL115), or both (MFL116), do not exhibit any growth in a minimal medium supplemented with 20 mM ethylene glycol ( FIG. 2A ). However, when the entire gcl cluster (gcl, hyi glxR, ttuD and pykF) is expressed as in strains MFL114 and MFL117, growth is observed. This reveals that expression of gcl alone is insufficient to support growth with ethylene glycol. Concomitant with growth, a rapid reduction of ethylene glycol concentrations is observed ( FIG. 2B ). Despite the fact that growth is not observed for KT2440, or the plasmid bearing variants MFL113, MFL115, and MFL116, partial metabolism of ethylene glycol in the first 24 h of incubation is observed for these cultures, although with varying efficiencies.

Construction of Genomically Engineered P. putida KT2440

To ascertain which genes are critical for ethylene glycol metabolism and to provide a base strain for further improvements, different combinations of genes from the gcl cluster were overexpressed in an intergenic region between fpvA and PP_4218 ( FIG. 3 ). The tac promoter was used for driving expression of the genes and the RBS in front of gcl was modified for optimal ribosome binding using an RBS calculator. Strain MFL168 includes all five genes (gcl, hyi, glxR, ttuD, and pykF), MFL170 includes three genes (gcl, hyi, glxR), MFL188 includes only two genes (gcl, glxR), representing the minimal requirement for a glyoxylate metabolizing unit. The genes behind gcl and the intergenic regions on the clusters were not genetically modified from that on the chromosome, except for the RBS of glxR in MFL188, which was again optimized using a RBS calculator.

Expression Analysis of Gcl Gene Cluster by qRT-PCR

As mentioned, the genes engineered into MFL168 were predicted to be transcribed in three different transcriptional units, namely gcl-hyi, glxR, and ttuD pykF. To resolve the question of whether genes located together in this gene cluster are co-expressed, we conducted quantitative real-time PCR experiments to measure transcript levels of gcl, hyi, glxR, ttuD, and pykF. We employed the housekeeping gene, rpoD, to quantify transcript levels between each of our samples using the 2 −ΔΔCt method.

As shown below, Ct values obtained for each sample and gene are provided in Table 5 and summarized as fold expression to transcript levels obtained from the control ( P. putida KT2440) after normalizing gene expression to rpoD ( FIG. 4 ).

Transcript levels of all gcl cluster genes in the wild type were very low. Values of 2−ΔCt of gcl, hyi, glxR, ttuD and pykF are 31-532 fold lower than for the control, rpoD (Table 5), confirming that this strain fails to induce this pathway on ethylene glycol. Transcript levels for gcl were approximately 2,000-fold higher in the engineered strains compared to the wild type, since it is driven by the strong tac promoter. For MFL168, transcript levels of the following genes in the cluster (hyi, glxR, ttuD, and pykF) are approximately the same, but 6-12 fold lower than gcl.

This was unexpected since a 27-base pair region (CCCTGTGGGAGCGGGCTCGCCCGCGAA (SEQ ID NO: 51)) which is present downstream from hyi is repeated as inverse complement further downstream, forming a 91 bp inverted repeat. Interestingly, a similar repeat region is present in the vicinity of gcl, downstream from PP_4296 (hypothetical protein) that differs by 1 nucleotide from the repeat downstream of hyi and forms an 81 bp inverted repeat. The presence of the inverted repeat did obviously not diminish the expression of downstream genes, since transcript levels for gcl, hyi, and glxR in MFL170 and MFL188 are similar to those in MFL168, for genes that were overexpressed. These results indicate that under the conditions tested, the putative terminators in the gcl gene cluster do not affect transcription of downstream genes, and that all of the genes in the cluster are transcribed as a single transcriptional unit and will thus be referred to as an operon.

Hydroxypyruvate Reductase Activity Assays

If a secondary pathway from tartronate semialdehyde to glycerate exists as shown in FIG. 1 , then a question remains as to which enzyme is responsible for directing hydroxypyruvate conversion to glycerate and back to the central metabolic pathway. From growth experiments and our qRT-PCR analysis, neither ttuD nor PP_0762 (hprA), a gene annotated as a hydroxypyruvate reductase in the BioCYC Database collection, are required for ethylene glycol assimilation ( FIG. 4 and Table 5). To identify the corresponding gene that is responsible for this catalytic step, we tested cell extracts from different strains for activity on the substrate hydroxypyruvate by monitoring NADH and NADPH oxidation ( FIG. 5 ). From these experiments, we found that cell extracts from P. putida KT2440 exhibit very little activity towards hydroxypyruvate, whereas, cell extracts from MFL168, MFL170, and MFL188 were all much more active for NADH oxidation in the presence of hydroxypyruvate. MFL188 only expresses two genes in addition to the control strains: gcl and glxR. Since Gcl does not have cofactor reducing capabilities, GlxR must be responsible for the activity.

Characterization of Engineered P. putida KT2440

To characterize the relative contribution of single genes of the gcl operon, the engineered strains were compared in minimal medium containing 20 and 50 mM ethylene glycol ( FIG. 6 ). On 20 mM ethylene glycol, both MFL168 (full operon) and MFL170 (gcl, hyi, glxR) grew sufficiently well consuming all substrate within 14 h ( FIG. 6B ). MFL188 (gcl, glxR) grew much more slowly and exhibited a long intermittent lag phase after an initial period of growth, although ethylene glycol was metabolized completely by MFL188 within 48 h. Growth was resumed at 72-92 h, after glycolate was consumed. There was transient accumulation of glycolaldehyde with very little production of glyoxal ( FIGS. 6C , D and E) while glycolate levels were elevated. MFL168 tended to perform better than the other strains when cultured in 50 mM ethylene glycol ( FIG. 6F-J ), however, there was some discrepancy between the duplicate flasks, whereby one culture consumed ethylene glycol sooner. MFL170 exhibited a two-staged growth associated with the accumulation of intermediates growing in 50 mM ethylene glycol ( FIG. 6F-J ), whereas MFL188 hardly grew in this higher substrate concentration, and the accumulated glycolate and glycolaldehyde were not metabolized. Intermediate glycolate levels ( FIG. 6H ) were higher in cultures with 50 mM, compared to 20 mM ethylene glycol, as expected. Glycolaldehyde levels rose to nearly 2.5 mM for both MFL168 and MFL170, but then fell as glycolate was consumed. Glyoxal was also present in culture samples and is derived from the oxidation of glycolaldehyde. In general, after an initial growth phase, further growth seems to be inhibited by the accumulation of ethylene glycol oxidation products. The engineered strains differ mostly in their metabolism of these intermediates, thereby recovering growth, especially at higher substrate concentrations. In particular, the aldehydes are highly toxic to microorganisms.

Therefore, an investigation of the intermediate metabolites (glycolaldehyde, glyoxylate, glycolate, oxalate, and glyoxal) and the substrate, ethylene glycol, was conducted to understand their impact on ethylene glycol metabolism.

Substrate, Metabolite, and Toxicity Assays

The toxicity of ethylene glycol, glyoxylate, glycolaldehyde, glyoxal, and oxalate to P. putida KT2440 were examined by monitoring growth in the Bioscreen C instrument (a microplate reader that monitors turbidity over time) in the presence of M9 minimal medium containing 20 mM glucose and the potential inhibitor. The average of at least five wells is shown in FIG. 7 . Ethylene glycol, sodium glyoxylate, and sodium glycolate at concentrations up to 100 mM are not significantly inhibitory, nor is sodium oxalate up to 50 mM. However, glycolaldehyde is toxic and completely inhibited growth at 4 mM. Glyoxal is also inhibitory to P. putida KT2440 at 5 mM, leading to a long lag phase before some growth can be observed. At 7.5 mM, glyoxal is lethal. Collectively, these data reveal that glycolaldehyde and glyoxal are the key intermediate metabolites that likely inhibit growth of P. putida on ethylene glycol. It has been reported that glycolaldehyde exerts a combinational inhibitory effect with other aldehydes. Thus, we investigated combinational effects of glycolaldehyde and glyoxal (Table 3 and FIG. 13 ). Growth rates in the presence of 2 mM glycolaldehyde or glyoxal are 0.22 h −1 and 0.25 h −1 , respectively. When combining 1 mM glycolaldehyde and 1 mM glyoxal, the growth rate is only 0.19 h −1 , demonstrating a minor synergistic effect on inhibition of the two compounds.

Characterization of a Glycolate Oxidase Overexpressing Strain

We observed that all three engineered strains transiently accumulated large amounts of glycolate in 20 mM ethylene glycol cultures ( FIG. 8 ). Therefore, glycolate oxidase was overexpressed in P. putida KT2440 by introducing glcDEF under control of the tac promoter (see FIG. 3 for details). The new strain (MFL185) was compared to MFL168 and the wild type on several different concentrations of ethylene glycol. Overexpression of the glycolate oxidase alone (MFL186) does not permit growth on ethylene glycol ( FIG. 8 ). However, the combined overexpression of glycolate oxidase and the gcl operon increased the growth rate and biomass yield on ethylene glycol with very little glycolate accumulation and no discernible glycolaldehyde present.

Growth rates, maximum dry cell weights, and consumed substrate are shown in Table 6 for cultures grown in the presence of 50 mM ethylene glycol which demonstrates a trend in ethylene glycol metabolic efficiencies for the engineered strains (MFL185>>MFL168>MFL170>MFL188).

We also compared growth on ethylene glycol at concentrations much higher than 50 mM, first in the Bioscreen C and then for MFL185 in shake flasks ( FIG. 9 ). Growth of MFL168 was optimal at 40 mM, reduced at 60 mM, and inhibited at 80 mM after a short initial growth. In contrast, MFL185, overexpressing the additional glycolate oxidase, grew in concentrations up to 2 M, the equivalent of 124 g/L. In shake flasks containing M9 minimal medium with 0.5 and 1.0 M ethylene glycol ( FIG. 9C-D ), MFL185 was able to completely consume 500 mM ethylene glycol within 120 h, although its growth lags compared to the culture with 250 mM. MFL185 consumed about 40% of the ethylene glycol at 1 M in shake flasks. There was some accumulation of glycolate (8-18 mM) between 14 and 24 h from samples grown in 100 to 500 mM samples; however, there was very little glycolaldehyde or glyoxal present. Due to high cell densities, we considered that perhaps nutrients might be limiting. To test this hypothesis, we added twice the M9 salts, which includes additional magnesium, nitrogen, calcium and iron and increased the inoculum from OD 600 =0.1 To 0.5. Ethylene glycol consumption was improved at 1 M (dashed line in FIG. 9C ) implying nutrients were indeed limited.

Production of mcl-PHAs from Ethylene Glycol

As a proof-of-concept for converting ethylene glycol to value-added products, we evaluated the ability of MFL185 to convert ethylene glycol into native carbon storage products, mcl-PHAs. Given that P. putida induces mcl-PHA production under nitrogen-limiting conditions, we grew cells in nitrogen-limiting M9 medium supplemented with 100 mM of ethylene glycol as the sole carbon source ( FIG. 11A ). We observed formation of mcl-PHAs from ethylene glycol by MFL185 using Nile Red staining ( FIG. 10A ), and monitored mcl-PHA production via flow cytometry over time. Cells reached maximum mcl-PHA production after 72 hours ( FIG. 11 B-C). MFL185 produced 52.8±2.2% of its dry cell weight as mcl-PHA at a product yield of 0.06 g of mcl-PHA produced per g of ethylene glycol consumed ( FIG. 10B ). Compositional analysis revealed that MFL185 produced the expected mcl-PHA chain length distribution in P. putida KT2440 which includes carbon chain lengths of C8, C10, C12, and C14, with greater than 93% of product belonging to C8 and C10 mcl-PHAs ( FIG. 10C ). Of note, the wild-type P. putida KT2440 strain is unable to grow or produce mcl-PHAs in nitrogen-limiting M9 medium containing 100 mM ethylene glycol ( FIG. 11A-C ). Comparison of mcl-PHAs production parameters of MFL185 revealed that mcl-PHA production from acetate and ethylene glycol are similar (yields: 0.05 per g of acetate vs 0.06 per g of ethylene glycol, 0.05>p) ( FIG. 10B-C ). This finding highlights that ethylene glycol is a suitable substrate for the production of PHAs using engineered P. putida strains as disclosed herein, compared to other C2 molecules. Collectively, these results demonstrate that MFL185 efficiently diverts ethylene glycol into an exemplary high-value product.

Although P. putida KT2440 has the genes necessary to convert ethylene glycol into cellular biomass, previous studies demonstrated that the organism is not capable of growing with ethylene glycol as the sole source of carbon and energy. We initially hypothesized that growth on ethylene glycol should depend on the functional expression of the gcl operon with glyoxylate carboligase (gcl) and tartronate semialdehyde reductase (glxR) as key enzymes. A prediction of the exact composition of a gcl operon from the genomic context was however, not straightforward. From a functional perspective, the co-transcription of gcl, hyi (hydroxypyruvate isomerase), glxR, ttuD, and pykF (pyruvate kinase) would make sense since those enzymes would allow two different routes for the conversion of glyoxylate to glycerate ( FIG. 1 ). However, from computational analysis, according to DOOR prediction ( FIG. 12 ), these genes are predicted to be transcribed in three different transcriptional units, namely gcl-hyi, glxR, and ttuD-pykF. As such, we conducted different qRT-PCR experiments and demonstrated, contradictory to the bioinformatics prediction, that all five genes are expressed as a single transcript representing one functional operon.

Without being limited by theory, the minimal requirement for glyoxylate metabolism from the gcl operon ( FIG. 1 ) is the expression of two genes (gcl, glxR). Notably, the expression of these genes in MFL188 enabled ethylene glycol metabolism, but at slower uptake rates than MFL170 that contains hydroxypyruvate isomerase (hyi) as an additional gene. Our results indicate that the gene ttuD is not necessary for growth in ethylene glycol since growth is achieved with the expression of gcl-glxR. However, its expression along with PykF, in MFL168, improved metabolic performance in shake flasks, compared to MFL170. Our biochemical data found no increased hydroxypyruvate reductase activity in strain MFL168 compared to MFL170, lacking ttuD, which was annotated as hydroxypyruvate reductase (BioCYC Database collection). However, in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database, TtuD is identified as a glycerate kinase. Our results suggest the latter is more likely. As such, we propose that TtuD represents a functional glycerate kinase and adjusted the pathway map accordingly ( FIG. 1 ). With activity assays using cell free extracts of MFL188, we demonstrated that GlxR could also function as a hydroxypyruvate reductase, in addition to its ability to convert tartronate semialdehyde to glycerate linking hydroxypyruvate to the central metabolic pathway ( FIG. 1 ). These results support that the activity of GlxR from P. putida using the substrates, tartronate semialdehyde and hydroxypyruvate, is in the presence of reduced pyridine nucleotide cofactors. Hydroxypyruvate functioned as a substrate at a 10-fold lower maximal velocity than tartronate semialdehyde. Also noted, was that glycolate inhibits this enzyme with a K i =3 mM. Therefore, an accumulation of glycolate could impede glyoxylate metabolism, which explains our observation that concentrations of glycolate were higher in strain MFL188 when grown in 20 mM ethylene glycol ( FIG. 6C ).

At elevated levels of ethylene glycol (>50 mM), glycolate and more importantly, glycolaldehyde and glyoxal levels increased to inhibitory levels, except in strain MFL185. The biomass yield for MFL168 in 20 mM ethylene glycol is high at 0.54 g DCW/g of ethylene glycol consumed and dropped to 0.16-0.27 g DCW per g of ethylene glycol consumed, when the substrate concentration was raised to 50 mM. The loss in biomass yield could be the result of the accumulation of intermediates, such as glycolaldehyde and glyoxal, which was not observed in 20 mM ethylene glycol ( FIG. 6 ), that might be diverted to other pathways that do not result in biomass formation. In strain MFL 185, the biomass yield in 50 mM ethylene glycol is 0.35 g/g, which is higher than from MFL168, but still lower at about 50% less than when grown in 20 mM ethylene glycol. It is also possible that the detoxification of glycolaldehyde and glyoxal requires ATP and NAD(P)H, in which carbon might be diverted towards ATP and redox cofactor generation rather than to biomass formation. In addition, higher glyoxylate concentrations result in some of it being redirected towards the glyoxylate shunt, which would result loss of carbon.

To improve the ethylene glycol conversion at higher substrate concentrations, we used the overexpression of glycolate oxidase to increase its conversion and thus to minimize the accumulation of the toxic intermediates glycolaldehyde and glyoxal. By combining glycolate oxidase with the gcl operon overexpression we generated a strain (MFL185) that can efficiently consume 0.5 M ethylene glycol (32 g/L) under shake flask conditions. Furthermore, we show that MFL185 can tolerate growth in up to 2 M (124 g/L) ethylene glycol. Moreover, with the addition of twice the M9 medium salt composition, we observed further consumption of ethylene glycol in the presence of 1 M (62 g/L) ethylene glycol (up to 37 g/L). Thus, with proper bioreactor control and the addition of limiting nutrients (i.e. nitrogen, iron) even higher substrate utilization might be possible.

Dynamic branching of intracellular metabolites is crucial for eliminating imbalance of cellular metabolism in microorganisms. For instance, in trehalose cycling, a side-pathway pushes glycolysis toward the trehalose metabolism for establishing steady state of the upper and lower pathway of glycolysis, thus eliminating the accumulation of intermediate metabolites. The failure to do so results in metabolic malfunctioning and growth arrest in high glucose containing medium. Similarly, Hyi siphons the ethylene glycol metabolite intermediate tartronate semialdehyde into hydroxypyruvate. Overexpression of hyi may facilitate a synthetic steady-state of ethylene glycol metabolism and relieve the bottleneck at tartronate semialdehyde, allowing for more efficient utilization of ethylene glycol. Otherwise cells exhibit metabolic and growth arrest. This might explain why the strains MFL168 and MFL170 perform better than strain MFL188. In addition, as described in the “push-and-pull” concept, the amplification of upstream, metabolite-forming pathways combined with a similar increase in the flux of downstream, metabolite utilization pathways could overcome feedback inhibition, and steer P. putida to achieve large flux of ethylene glycol at high rate.

Beyond the superior growth characteristics of MFL185 in ethylene glycol, we demonstrated that ethylene glycol could be converted to high-value products such as mcl-PHAs. Together with the conversion of terephthalate, this now enables the complete biotransformation of depolymerized PET into mcl-PHAs with P. putida . mcl-PHAs can be upgraded into chemical precursors and fuels via straightforward catalytic process. Several metabolic engineering strategies have been developed to enhance mcl-PHAs production in P. putida , and these approaches could be used to increased mcl-PHA production in the MFL185 strain. As disclosed herein, metabolic modeling coupled with techno-economic analysis are useful tools for identifying ideal product(s) from ethylene glycol. The source of ethylene glycol should be considered for tailoring MFL185 as a biocatalyst to valorize ethylene glycol containing streams. For instance, in PET-degraded streams, ethylene glycol could be used for growth, energy, and/or PHA production.

Thus, presented herein is an engineered strain (MFL185) for ethylene glycol consumption, and a foundation strain for further development as a biocatalyst for the conversion of ethylene glycol in waste plastics streams, and for the conversion of glycolaldehyde in thermochemical wastewater streams, and generally for additional environmental bioremediation applications.

Plasmid Construction of pMFL113-pMFL117

The plasmid, pBTL2 was amplified with primers as designated above to introduce NotI and SbfI sites or PacI and SbfI sites to the plasmid. PCR products obtained from amplification from KT2440 genomic DNA using primers above and the vector were purified and digested with restriction enzymes noted above, purified again and ligated together, then transformed into NEB 5-alpha F′Iq E. coli . After confirmation of sequence, DNA was transformed into P. putida KT2440 and the strains designated as MFL113 to MFL117.

Plasmid construction details of pMFL160, pMFL161, pLJ030, pLJ031 and pLJ032 (Integration constructs for MFL168, MFL170, MFL185, MFL186 and MFL188) are depicted in Table 8.

In order to construct the integration plasmids, pMFL160 and MFL161, 3 PCR fragments for assembly #1 (Table 8) were assembled using NEBuilder® HiFi DNA Assembly Master Mix following manufacturer's instructions. The fragment was digested with XbaI and SpeI along with PCR fragments generated from oMFL276 and oMFL279 (gcl-hyi-glxR) or oMFL276 with oMFL280 (gcl operon), which were also cut with XbaI, and SpeI. Fragments were PCR purified, ligated and transformed into E. coli as described above to generate either pMFL160 (integration overexpression construct for MFL170 (gcl-hyi-glxR)) or to generate pMFL161 (integration construct for MFL168 and MFL185 expressing the entire gcl operon), both of which are contain the pK18mobsacb backbone. The integration plasmid, pMFL160 was used as a template for the construction of pJL032 for integration of gcl-glxR. Briefly, three fragments were assembled as shown in the table above, then assembled (assembly #2) and transformed into NEB 5-alpha F′Iq E. coli . For assembly of the integration construction to insert the tac promoter in front of the glcDEF operon, three PCR fragments were generated (assembly #3 above), assembled and transformed into NEB 5-alpha F′Iq E. coli . All plasmids were confirmed by sequencing prior to integration into P. putida KT2440.

Redox Equivalent Homeostasis in the Utilization of Ethylene Glycol as Co-Substrate

An analysis of the P. putida genome reveals that it possesses open reading frames that putatively encode enzymes that if expressed might be useful in metabolic pathways that could potentially enable ethylene glycol metabolism. In one such ethylene glycol metabolic pathway, referred to herein as the “energy yielding pathway”, the diol (ethylene glycol) is converted into glyoxylate in a series of oxidation reactions catalyzed by a set of redundant dehydrogenases, PQQ-dependent PedI, PedE and PedH (see FIG. 1 ). Further oxidation from glyoxylate to oxalate can occur, but only very small traces of this metabolically dead-end product have been observed.

The complete conversion of ethylene glycol to glyoxylate yields three reducing equivalents, either in the form of NADH, PQQH 2 , or a direct coupling to the electron transport chain. Glyoxylate can be further metabolized by the AceA or GlcB enzymes involved in the glyoxylate shunt. Although this shunt is usually a carbon conservation pathway for growth on C2 substrates that enter primary metabolism at the level of acetyl-CoA, the overall stoichiometry of glyoxylate metabolism via either of the reactions catalyzed by the AceA or GlcB enzymes can only yield 2 CO 2 and 2 reducing equivalents (see FIG. 1 ). Thus, the overall conversion of ethylene glycol to 2 CO 2 can yield a total of five reducing equivalents, making it a promising energy-yielding co-substrate. In comparison, typical redox energy co-substrates such as formate can only yield one reducing equivalents per C mole of substrate, and even the complete oxidation of glucose through primary metabolism only yields approximately 1.66 reducing equivalents per C mole of substrate.

To investigate the applicability of ethylene glycol as a co-substrate, P. putida KT2440 was cultured in carbon-limited chemostat cultivations with acetate as carbon source, and either ethylene glycol or glyoxylate as an energy source. Acetate was chosen because it induces the glyoxylate shunt enzymes. Compared to the control with only acetate, a co-feed of ethylene glycol or glyoxylate significantly increased the biomass yield on acetate by 29.6±1.1% or 22.2±8.2%, respectively. This increase can likely be attributed to the additional reducing equivalents generated through the co-substrate metabolism, enabling a more efficient carbon metabolism of the primary substrate. However, the fed ethylene glycol was only partly metabolized, and several intermediate oxidation products were secreted. A limitation in the upstream oxidation reactions can be excluded, since glyoxylate was also not completely metabolized under these conditions.

Energy and redox equivalent homeostasis is essential for efficient growth of a bacterial cell. As such, the observed inefficiency in metabolism of ethylene glycol could result from a potential imbalance in the pool of redox equivalents. Assuming that all glyoxylate is metabolized through the glyoxylate shunt, 27.3±2.6 mmol (g CDW) −1 h −1 of reducing equivalents were generated through the co-metabolism of ethylene glycol. In contrast, under similar conditions reached the maximal achievable biomass yield on glucose already with 7.6±0.9 mmol (g CDW) −1 h −1 using a co-feed of formate. Thus, under these conditions, the generated flux of reducing equivalents from an ethylene glycol co-feed likely constitute a strong surplus in energy. This is further supported by the fact that a glyoxylate co-feed, yielding only 14.4±0.5 mmol (g CDW) −1 h −1 of reducing equivalents, enabled almost the same biomass yield increase as the ethylene glycol co-feed. This surplus of reducing equivalents leads to an imbalance of redox cofactors, inhibiting the further conversion of ethylene glycol under these conditions.

Isolation of Mutants Able to Utilize Ethylene Glycol as Sole Carbon Source

In an alternative to the energy yielding pathway, the genome of P. putida KT2440 contains open reading frames (PP_4297-PP_4301) that if expressed might encode for enzymes useful in a pathway to metabolize glyoxylate through a glyoxylate carboligase which converts two glyoxylate into tartronate semialdehyde and CO 2 . In this pathway, glyoxylate is converted to glycerate, either directly or via hydroxypyruvate, and subsequently to 2-phosphoglycerate (see FIG. 1B ). Thus, this pathway is referred to herein as the ‘gel pathway’. If operative, the gcl pathway could allow for the utilization of ethylene glycol as a sole carbon source.

Adaptive laboratory evolution (ALE) techniques were applied to P. putida KT2440 and lead to the emergence of adaptive mutations allowing for growth on ethylene glycol as a sole carbon source. Two independent ALE experiments were performed in two different laboratory P. putida KT2440 wildtype strains using different minimal media recipes supplemented with ethylene glycol as a sole carbon source. As depicted in FIG. 19 , as a result of the ALE experiments, adaptive mutants emerged, leading to observable growth after a lag phase of 4-6 days.

Three isolates were obtained from independently evolved cultures directly after this initial growth. The best growing clones from each culture were selected from nine individual clones and subcultured three times on LB-agar plates to obtain strains E1.1, E1.2 and E1.3.

A series of three parallel ALE cultivations was performed where batches were sequentially re-inoculated into fresh medium with ethylene glycol after growth became apparent by visual inspection, see FIG. 17 . After six serial transfers, 36 individual strains were isolated on LB-agar plates and grown in liquid cultures. The best growing strains were selected to obtain strains E6.1 and E6.2.

All five resulting strains (E1.1, E1.2, E1.3, E6.1, and E6.2) demonstrated a stable ethylene glycol-growing phenotype. No major differences could be observed within the E1 and E6 groups. When grown in minimal medium with 26.7±0.4 mM ethylene glycol as a sole carbon source, both groups grew at approximately the same initial rate (0.19±0.02 h −1 ). However, the maximum biomass concentration of the E6 cultures (0.63±0.02 g cdw L −1 ) was significantly higher than that of the E1 cultures (0.49±0.07 g cdw L −1 ) ( FIGS. 15A, 15C ). The differences between the E1 and E6 strains becomes more apparent upon cultivation in 120 mM ethylene glycol ( FIG. 15D ). In this case, the E6 strains grew 1.4-fold faster than the E1 strains (E1: 0.08±0.004 h −1 , E6: 0.12±0.004 h −1 ), also reaching a higher final biomass concentration. The difference between E1 and E6 groups was also reflected in the metabolism of ethylene glycol and the formation of intermediate oxidation products. As depicted in FIG. 15B , the ethylene glycol uptake rate of E6.1 is about 1.8-fold higher than that of E1.1. E6.1 also produces up to 7.9-fold more glycolate than E1.1.

Genomic and Metabolic Context of Adaptive Mutations

Whole genome resequencing was performed on the E1 and E6 strains in order to discover the mutations responsible for the stable phenotypic switch in the E1 and E6 ALE strains. In comparison to publicly available sequence of P. putida KT2440 (AE015451.2) (see FIG. 12 ), the sequences of E1 and E6 had 92-99 Single Nucleotide Polymorphisms (SNP) and Insertion-Deletion polymorphisms (InDel). However, the vast majority of these were already present in the two wildtype strains from which E1 and E6 were derived. The mutations were mainly in non-coding regions, due transposon effects, or errors due to low coverage, read quality, or silent mutations having no major effect.

The mutations identified in strains E1 and E6 were very similar to each other, even though they were derived from different wildtype P. putida KT2440 strains. After subtracting parental, silent, and intergenic mutations, two additional mutated regions were identified in the E1 and E6 strains. One region (coordinates 4866804 to 4902814, of P. putida KT2440) was mutated in all the evolved strains (E1.1, E1.2, E1.3, E6.1, and E6.2), while the other region (coordinates 2325342 to 2334253) was only mutated in the E6 group (E6.1, and E6.2), see FIG. 16 .

In the first region, E1.1, E1.2, E1.3, and E6.1 contained mutations in the gene with locus tag PP_4283 encoding a putative GntR-type transcriptional regulator gclR. Using RegPrecise, GclR is predicted to be a regulator of xanthine metabolism, with two predicted binding sites upstream of the gcl gene. The mutations in the first region included one nonsense mutation in E6.1 giving rise to a stop codon in the 4th triplet, indicating that the gene function is disrupted. Strain E6.2 did not contain a mutation in gclR, instead it has a SNP 12.5 kb downstream in the promotor region of the gcl gene, which is the first gene in the PP_4297-PP_4301 cluster that encodes the enzymes of the gcl pathway.

Transcript levels of all five genes (PP_4297-PP_4301) in this cluster are very low in the wildtype P. putida KT2440 strain. In contrast, all five genes (PP_4297-PP_4301) in the cluster were strongly upregulated in strains E1.1, E6.1 and E6.2 having 2ΔΔCt values between 71 and 842 vs. wildtype, see FIG. 4D . The distally located hprA gene (PP_0762), which encodes a second possible glyoxylate/hydroxypyruvate reductase, is not expressed under these conditions, see FIG. 16D . Thus, GclR is a repressor of the PP_4297-PP_4301 gene cluster. Through the disruption of gclR, or the disruption of the GclR binding site in the case of strain E6.2, the repression of the transcription and expression of all five genes (PP_4297-PP_4301) in the cluster was substantially eliminated, thereby enabling growth on ethylene glycol as a sole carbon source.

In P. putida , ethylene glycol and/or glyoxylate are not the effectors which bind GclR to relieve repression of the PP_4297-PP_4301 gene cluster. The genomic context of the gclR gene in P. putida KT2440 which, similarly to other organisms such as E. coli, Streptomyces coelicolor , and Bacillus subtilis , encode multiple genes known or predicted to be involved in the metabolism of purines via allantoin and glyoxylate, see FIG. 10 . In aerobically growing E. coli , the genes encoding the gcl pathway are repressed in the presence of allantoin through the action of the AllR regulator. This repression is alleviated by glyoxylate, which concomitantly induces an alternative allantoin metabolic pathway which ultimately yields ammonia, CO 2 and ATP, predominantly active under anaerobic conditions. This alternative pathway seems to be absent in P. putida KT2440, befitting its obligate aerobic lifestyle. The genomic context of gclR suggests that the failure of wildtype P. putida KT2440 to activate the gcl pathway on ethylene glycol or glyoxylate is because it is part of a larger metabolic context, governed by inducers that lie upstream of their metabolism.

Both wildtype P. putida KT2440 and strain E6.1 are able to grow on allantoin as a sole carbon and nitrogen source (see FIG. 17 ), showing that allantoin, and not glyoxylate, is the inducer of the genes encoding the gcl pathway. A co-feed of allantoin and ethylene glycol resulted in a higher biomass concentration through the activation of the gcl pathway. Similar results were obtained with xanthine, which also enabled growth on ethylene glycol.

E6.2 was not able to utilize allantoin as sole nitrogen source, although it retained the ability to utilize it as a carbon source, either with or without the addition of ethylene glycol. This indicates that there is regulatory cross-talk between allantoin and ethylene glycol metabolism, possibly involving the PP_4296 gene whose expression is likely affected by its mutation in E6.2. The product of the PP_4296 gene shows sequence similarity to the E. coli GlcG protein which is implicated in glycolate metabolism.

In addition to the mutations involving gclR, an additional mutation was found in both of the strains E6.1 and E6.2. Both strains contained the same missense mutation (E34G) in the gene with locus tag PP_2046, a LysR-type transcriptional regulator. This regulator controls the adjacent operon which encodes a beta-oxidation pathway including a CoA-transferase, an acyl-CoA thioase, and several (enoyl-CoA-) dehydrogenases, see FIG. 16B . Although the C2 compound ethylene glycol cannot be metabolized through beta-oxidation, the dehydrogenases could accelerate its oxidation as exemplified in that E6.1 transiently accumulates much more glycolate than E1.1, see FIG. 3B . However, initial ethylene glycol oxidations are mostly performed in the periplasm by PedE and/or PedH since a knockout of the pedE-pedI cluster (PP_2673-PP_2780) in the E6 strains eliminates the ability to grow on ethylene glycol. CoA activation may be involved and enzymatic activities in the operon suggest that glycolyl-CoA is formed and converted to glyoxyl-CoA and subsequently to glyoxylate. An additional pathway would help to accelerate the apparent rate-limiting step in the E6 mutants, and possibly also alleviate the toxicity of glyoxylate by CoA activation (see FIG. 20 ).

Reversed Engineering of Ethylene Glycol Metabolism

Mutations in the gclR and PP_2046 genes contribute to the efficient growth of P. putida KT2440 on ethylene glycol as a sole carbon source. To determine how these mutations assert their effect, we replicated the phenotype of the mutant strains through reverse engineering.

Growth on ethylene glycol was assessed in gclR knockouts of the wildtype, E6.1, and E6.2 strains, see FIG. 17 . P. putida KT2440 ΔgclR grows readily on minimal medium with ethylene glycol as the sole carbon source, while the growth of strain E6.1 ΔgclR is not significantly changed compared to its progenitor E6.1. Thus, the gclR mutations in the ALE strains were disruptive in nature, and GclR is a repressor of the genes encoding the gcl pathway.

Strain E6.2 ΔgclR grows slower than its parental strain. This, together with the fact that E6.2 can't use allantoin as sole nitrogen source, is indicative of a more complex regulatory mechanism, perhaps one analogous to that of E. coli 's A11R, which can bind both allantoin and glyoxylate leading to either the repression or the activation of genes. To verify that it is only the activation of the gcl pathway, excluding any polar regulatory effects, the gcl and glxR genes were expressed episomally in wildtype P. putida KT2440 under the control of the LacIq-Ptrc promoter on plasmid pSEVA234 (SEQ ID NO: 5). Indeed, the resulting strain KT2440 pSEVA234_gcl-glxR grows on ethylene glycol. Although qRT-PCR experiments determined that in the ALE strains, the five genes were upregulated, two of these are redundant, and P. putida KT2440 contains another gene encoding a glycerate kinase (PP_3178). In an embodiment, only gcl and glxR need to be expressed to enable growth on ethylene glycol. A significant contribution of the redundant pathway via hydroxypyruvate is indicated by strain KT2440 pSEVA234 gcl-glxR growing slower than KT2440 ΔgclR or the ALE strains.

In contrast to the ALE mutations in gclR, the E6 strains both contained the same SNP in the PP_2046 gene leading to an E34G substitution. It is unclear whether this mutation simply disrupts activity, or whether it changes the functionality of the encoded regulator. To distinguish between these effects, PP_2046 was knocked out and the mutant variant of the gene was episomally re-introduced into this knockout under the control of the NagR-P¬nagAa promoter on plasmid pBNTmcs. These modifications were made in P. putida KT2440 ΔgclR, because modification of PP_2046 alone didn't enable growth on ethylene glycol. Both strains P. putida KT2440 ΔgclRAPP_2046 pBNT_PP_2046E34G and the empty vector control KT2440 ΔgclRΔPP_2046 pBNTmcs still grow on ethylene glycol, but the latter much slower than the former.

Engineering P. putida KT2440 for Efficient Ethylene Glycol Utilization

To further expand the ability of P. putida for substrates of industrial interest, the metabolism of ethylene glycol was enhanced via metabolic engineering through the overexpression of existing regulated pathways that include the glyoxylate carboligase operon. Growth in concentrations of ethylene glycol above 50 mM was inhibited by the accumulation of toxic intermediates, glycolaldehyde and glyoxal. The additional overexpression of glycolate oxidase (glcDEF) operon removed the glycolate bottleneck and minimized the production of these toxic intermediates, permitting the growth in up to 2M (120 g/L) and consumption of greater than about 0.5M (30 g/L) of ethylene glycol.

Construction and Characterization of Integrated Engineered P. putida KT2440

In order to ascertain which genes were important for ethylene glycol metabolism and to provide a base strain for further improvements, genes of interest were integrated into an intergenic region between fpvA and PP_4218. This region was chosen because we have successfully integrated other genes in this location, in addition to having native terminators present in both directions. The tac promoter was used for driving expression of the operon and the RBS in front of gcl modified for optimal ribosome binding, see FIG. 3 . MFL168 included all 5 genes (gcl, hyi, glxR, PP_4300 and pykF) behind the tac promoter, MFL170 included 3 genes (gcl, hyi, glxR) MFL188 expressed only 2 genes (gcl, glxR), the minimal requirement for a glyoxylate metabolizing unit. The genes behind gcl and the intergenic regions on the operons were not genetically modified from that on the chromosome, except for the RBS of glxR in MFL188 that was optimized with the use of an RBS calculator.

Cultures were compared for growth in the presence of 20 mM ethylene glycol in minimal medium, see FIG. 21 . Both MFL168 (gcl, hyi, glxR, PP_4300 and pykF) and MFL170 (gcl, hyi, glxR) grew sufficiently well and were able to consume ethylene glycol within 14 hours, see FIG. 21B ). MFL188 expressing only gcl and glxR grew much more slowly and exhibited a long lag phase until growth was nearly complete at 90 hours, although ethylene glycol was metabolized completely within 48 hours, due to the incomplete utilization of glycolate and some glycolaldehyde that eventually dissipated until nearly gone at 90 hours, see FIGS. 21C and D).

Integrated strains were tested for growth in 50 mM ethylene glycol and it was discovered that strains had more difficulty in consuming the higher concentration of ethylene glycol, see FIG. 19 . MFL168 required 72 hours for total substrate utilization, and for MFL170, 96 hours, whereas MFL188 barely grew. Glycolate levels were much lower than that observed for 20 mM ethylene glycol, but glycolaldehyde measurements rose to nearly 2.5 mM for both MFL168 and MFL170. Glyoxal was also present in culture samples. Glyoxal is derived from the oxidation of glycolaldehyde. Glycolaldehyde is toxic and has been shown to be more inhibitory than 5-hydroxymethyl furfural (5-HMF) and furfural for Saccharomyces cerevesiae . Therefore, an investigation of glycolaldehyde toxicity as well as other intermediate metabolites (glyxoylate, glycolate, oxalate, and glyoxal) and the substrate, ethylene glycol, was conducted to better understand their impact on ethylene glycol metabolism.

Substrate, Metabolite, and Competitive Inhibition Toxicity Assays

The toxicity effects of ethylene glycol, glyoxylate, glycolaldehyde, glyoxal and oxalate on P. putida KT2440 were examined by monitoring growth in the Bioscreen C instrument (a microplate reader which monitors turbidity over time) in the presence of M9 minimal medium containing 20 mM glucose and the inhibitor, see FIG. 20 . The average OD of at least 5 wells was plotted in FIG. 9 . Ethylene glycol, sodium glyoxylate, and sodium glycolate at concentrations up to 100 mM were not significantly inhibitory; nor was sodium oxalate up to 50 mM. However, glycolaldehyde was very toxic and completely inhibited growth at 4 mM and inhibits growth at 3 mM. Glyoxal, an oxidation product of glycolaldehyde, is also very inhibitory to P. putida KT2440 growth at a 5 mM concentration where a long lag phase is observed. At 7.5 mM, glyoxal is lethal to the cells. Therefore, glycolaldehyde and glyoxal are intermediate metabolites which result in ethylene glycol toxicity on P. putida KT2440. Glycolaldehyde exerts combinational inhibitory effect with other compounds, such as 5-HMF, furfural, and methylglyoxal. The combinational inhibitory effect of glycolaldehyde and glyoxal was determined by running three level full-factorial growth experiments, see FIG. 13 and Table 3.

Glycolaldehyde and glyoxal have a synergistic inhibitory effect where, the growth rates in the presence of 2 mM glycolaldehyde is 0.291±0.001 h −1 , 2 mM glyoxal is 0.297±0.001 h −1 , and 1 mM glycolaldehyde+1 mM glyoxal is 0.197±0.001 h −1 .

Characterization of an Overexpressing Glycolate Oxidase Strain

In 20 mM ethylene glycol cultures, all three engineered strains accumulated large amounts of glycolate (see FIG. 21 ), particularly within the first 14 hours for MFL168 and for MFL170 and for MFL188, between 48 and 72 hours. An overexpressing glycolate oxidase was constructed by introducing a tac promoter in front of the native glycolate oxidase operon that includes 5 genes, 3 of them necessary for glycolate oxidase activity (glcDEF), see FIG. 3 . A comparison of the new strain (MFL185) with MFL168 and the wild-type strain, P. putida KT2440, was made in several different concentrations of ethylene glycol. FIG. 22 compares the strains when grown in 50 mM ethylene glycol. MFL185 grows much faster in ethylene glycol (top line in FIG. 22A ), consumes ethylene glycol faster, with very little glycolate accumulation and no discernible glycolaldehyde present.

Growth at concentrations higher than 50 mM was compared between Bioscreen C and in shake flasks, see FIG. 23 . FIG. 23A compares growth of MFL168 in up to 80 mM ethylene glycol. Growth was optimal at 40 mM, reduced at 60 mM and inhibited for growth at 80 mM. In contrast, MFL185, containing an additional overexpressing glycolate oxidase, grew in concentrations up to 1.5 M, the equivalent of 90 g/L. This is significantly higher than that achieved without the glycolate oxidase expression, approximately 25-fold higher.

A shake flask study in M9 minimal medium containing only ethylene glycol from 25 mM to 3 M was conducted to determine whether MFL185 can utilize this much extra ethylene glycol, see FIGS. 23C and D which depict concentrations of 100 mM to 1 M of ethylene glycol. There was some growth at 2 M in both the microplates and in the shake flasks but very little ethylene glycol used. MFL185 was able to completely consume 500 mM ethylene glycol within 120 hours, although its growth lagged behind growth in 250 mM ethylene glycol. MFL185 consumed about 40% of the ethylene glycol present in 1 M under these shake flask conditions. There was some accumulation of glycolate (from 8-18 mM) between 14 and 24 hours from samples grown in 100 to 500 mM samples, however, there was very little glycolaldehyde or glyoxal present.

qRT-PCR

The genes engineered into MFL168 were predicted to be transcribed in three different transcriptional units, namely gcl-hyi, glxR, and PP_4300-pykF. There is a strong inverted repeat located in the intergenic region between hyi and glxR, which indicates that glxR would be on a different transcript, however, no potential promoters were predicted to be present upstream of glxR using an online software prediction program, BPROM. Quantitative real-time PCR experiments were conducted to measure transcript levels of gcl, hyi, glxR, PP_4300 and pykF in addition to PP_0762 to determine whether genes located together on this operon are expressed. PP_0762 was annotated to function as a hydroxypyruvate reductase and could be involved in converting hydroxypyruvate to glycerate in ethylene glycol metabolism. A housekeeping gene, rpoD, was used to quantify transcript levels between each of our samples using the 2− ΔΔ Ct method. Cultures were grown up in M9 minimal medium with 40 mM sodium acetate and 20 mM ethylene glycol. For ethylene glycol consuming cultures, acetate was utilized first, followed by ethylene glycol. All cultures were harvested at about an OD 600 of 1 at which point, the substrates acetate and ethylene glycol were still available for growth.

Ct values were obtained for each sample and gene and are summarized as fold expression to transcript levels obtained from the control ( P. putida KT2440) after normalizing gene expression to rpoD, see FIG. 24 . Transcript levels for gcl were expected to be much higher than in the control strain, since it is driven by the strong tac promoter and are about 2,000 times greater in MFL168 than in the wild-type strain. For MFL168, transcript levels of both hyi and glxR are approximately the same and about 300 times greater than the control. Transcript levels for both PP_4300 and pykF are slightly lower but still much higher than the control (about 150-300) fold greater. Transcript levels for gcl, hyi and glxR in MFL170 and MFL188 are similar to those in MFL168, for genes that were overexpressed. These results indicate that all of these genes are indeed located on the same operon and are being expressed together. The transcript levels for PP_0762 (hprA) were also measured and had a relatively low expression in the control strain and in the engineered strains under these conditions. Under these conditions, it is unlikely that this enzyme plays a role in conversion of hydroxypyruvate to glycerate.

Activity Assays

Experiments were performed to determine which enzyme is responsible for converting hydroxypyruvate to glycerate. PP_4300 and hprA (PP_0762) are not required for ethylene glycol assimilation, so assays were conducted to test cell extracts for activity on the substrate hydroxypyruvate by monitoring NADH and NADPH oxidation, see FIG. 25 . Cell extracts from P. putida KT2440 exhibited very little activity towards hydroxypyruvate, whereas, cell extracts from MFL168, MFL170 and MFL188 were all positive for NADH oxidation in the presence of hydroxypyruvate. MFL188 expresses two genes above that of the control strains: gcl and glxR. Since Gcl does not have cofactor reducing capabilities, GlxR is likely responsible for the activity. Previous work purified and characterized tartronate semialdehyde reductase (GlxR) from P. putida and discovered that hydroxypyruvate also acts as a substrate, albeit at a 10-fold lower maximal velocity than when tartronate semialdehyde was used as a substrate. Furthermore, that work also noted that glycolate inhibits this enzyme with a K i =3 mM, which may explain why MFL188 does not perform as well as MFL168 or MFL170.

A prediction of the exact composition of a gcl operon from the genomic context is not straightforward. From a functional perspective the co-transcription of gcl, hyi (hydroxypyruvate isomerase), glxR (tartronate semialdehyde reductase) and PP_4300 (hydroxypyruvate reductase) and pykF would make functional sense since they would allow two different routes for the conversion of glyoxylate to glycerate, see FIG. 1 . However, from computational analysis, according to DOOR prediction, these genes are predicted to be transcribed in three different transcriptional units, namely gcl-hyi, glxR, and PP_4300-pykF. The qRT-PCR experiments performed herein indicate that they are expressed as one operon. In an embodiment, the minimal requirement for a glyoxylate-metabolizing unit is the expression of two genes (gcl, glxR), such as in MFL188, which does metabolize ethylene glycol, but at slower uptake rates than MFL170 that contains an additional gene (hyi). It was discovered with activity assays of cell extracts that GlxR could also function as a hydroxypyruvate reductase, converting hydroxypyruvate to glycerate which connects hydroxypyruvate to the central metabolic pathway, see FIG. 1 . Hydroxypyruvate can act as a substrate for GlxR and glycolate can inhibit its activity. This might explain why strains MFL168 and MFL170 perform better than MFL188.

The two additional enzymes, PP_4300 (TtuD) and PykF, expressed in MFL168, compared to MFL170, showed improved performance under our conditions, but not to a lesser extent than the addition of Hyi. PP_4300 was predicted to function as a hydroxypyruvate reductase (BioCYC Database collection), but PP_4300 was identified as a glycerate kinase in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database, see FIG. 1 .

At elevated levels of ethylene glycol (>50 mM), glycolate, glycolaldehyde and glyoxal levels increased. The accumulation of glycolate caused the concentration of glycolaldehyde to increase to inhibitory levels and is likely responsible for inhibition of GlxR activity. A strain (MFL185) was engineered to overexpress glycolate oxidase. MFL185 can metabolize 500 mM ethylene glycol (30 g/L) under shake flask conditions. MFL185 will tolerate growth in up to 1.5 M (90 g/L) ethylene glycol. Increased substrate utilization with proper fermentation control and the addition of limiting nutrients (i.e. nitrogen, iron) may be possible.

The deregulation of the gcl operon allows for growth of P. putida KT2440 in ethylene glycol. Furthermore, growth of engineered strains in higher concentrations of ethylene glycol can be inhibited by the accumulation of the toxic intermediates, glycolaldehyde and glyoxal. The addition of glycolate oxidase removes the glycolate bottleneck and minimizes the production of these toxic intermediates, allowing for the growth in up to 2 M (120 g/L) and consumption of 0.5M (30 g/L) or more of ethylene glycol under the shake flask growth conditions.

Plasmid Construction

Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers synthesized by Integrated DNA Technologies (IDT) were used in all PCR amplification for plasmid construction. Plasmids were constructed using Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer's instructions. Plasmids for gene integration were constructed in pK18mobsacB from ATCC (American Type Culture Collection, Manassas, Va.), which is unable to replicate in P. putida KT2440, and contains the kanamycin resistant marker to select for integration of the plasmid into the genome by homologous recombination and sacB to counter select for recombination of the plasmid out of the genome. The plasmid, CJ0XX, used for of integration of operons containing the gene gcl (MFL168, MFL170, MFL185, and MFL188) contains the 1 kb homology region on either side of the intergenic region immediately after the fpvA (outer membrane ferripyoverdine receptor) terminator and PP_4218 (lipase/esterase) of P. putida KT2440. Features include the tac promoter to drive operon expression and a tonB terminator situated behind the operon ( FIG. 3 ). The RBS for gcl was optimized for gcl expression and were the same for MFL168, MFL170 and MFL185. The RBS for glxR in MFL188 was also optimized. The remaining RBS and gene sequences remained the same as on the native chromosome. The overexpression of glycolate oxidase genes (glcDEF) in MFL185, MFL186 and MFL188 was achieved by integrating the tac promoter upstream of the glycolate oxidase operon (glcDEFG_PP_3749) and behind the native promoter, while additionally optimizing the RBS for glcD. Primers used for PCR amplification and Gibson assembly are listed in Table 4.

Plasmids were transformed into competent NEB 5-alpha F′Iq E. coli (New England Biolabs) according to the manufacturer's instructions. Transformants were selected on LB (Lennox) plates containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 15 g/L agar, supplemented with either 10 μg/mL tetracycline or 50 μg/mL kanamycin grown at 37° C. The sequences of all plasmid inserts were confirmed using Sanger sequencing (GENEWIZ, Inc.). Specific plasmid construction details can be found in the supplementary materials and methods.

Strain Construction

P. putida KT2440 (ATCC 47054) was used as the basis of strain engineering and gene replacements were made using the antibiotic/sacB system of selection and counter-selection. To prepare cells of P. putida KT2440 and strains derived from it for transformation by electroporation, cultures were grown up overnight in LB broth and incubated at 30° C., shaking at 225 rpm. The next day, cells were centrifuged 15,000 rpm in an Eppendorf centrifuge for 1 minute at room temperature, washed three times in 0.3M sucrose in half the original volume, centrifuged again then resuspended then resuspended in 1/50th of the culture's original volume in 0.3M sucrose. Cells were immediately used for electroporation by introducing 5 μL (200 ng-2 μg) of plasmid DNA to 50 μL of the electrocompetent cells, transferred to a chilled 0.1 cm electroporation cuvette, and electroporated at 1.6 kV, 25 μF, 200 ohms. 950 μL SOC outgrowth medium (NEB) was added to the cells immediately after electroporation and the resuspended cells were incubated shaking at 225 rpm, 30° C., for two hours. The entire transformation was plated on an LB agar plate containing appropriate antibiotics (50 μg/mL kanamycin for pBTL2 and pK18mobsacB-based plasmids) and incubated at 30° C. overnight. Transformants were restreaked for single colonies on LB agar and incubated at 30° C. overnight to reduce the possibility of untransformed cells being transferred. For sucrose counter-selection, restreaked transformants were streaked for single colonies on YT+25% sucrose plates (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, 18 g/L agar), and incubated at 30° C. overnight. P. putida KT2440 containing the sacB gene can grow, although very slowly, on YT+25% sucrose media. Therefore, colonies presumed to have recombined the sacB gene out of the genome—those colonies that were larger than most were restreaked on YT+25% sucrose plates and incubated at 30° C. overnight to reduce the possibility that cells that had not recombined would be carried along with sucrose resistant isolates. Colonies from the second YT+25% sucrose plates were subjected to colony PCR to check for gene replacement at both junctions. These isolates were also plated on LB plates containing appropriate antibiotics to ensure that they had lost antibiotic resistance and, thus, represented pure gene replacements. FIG. 3 depicts a schematic representation of the organization of the open reading frames of some of the engineered strains produced in this work. For overexpression, the tac promoter was used and ribosomal binding sites were optimized using an online RBS calculator.

Toxicity Tests in Bioscreen C

Growth curves were obtained from Bioscreen C analyzers (Growth Curves US, Piscataway, N.J.). Overnight cultures of P. putida KT2440 were grown in M9 medium containing 20 mM glucose starting at an OD 600 of 0.05-0.1, 30° C., 225 rpm (baffled shake flask) until the OD 600 reached ˜1.0-1.5. Cells were concentrated by centrifugation and inoculated into wells of Bioscreen C microplates containing various inhibitors and concentrations in the presence of 20 mM glucose at and additional OD 600 =0.05 in a total volume of 300 μL. Incubations were performed at 30° C. and absorbance readings were taken every 15 min. Operation of the Bioscreen C and collection of turbidity measurements (OD 420-580 ) were computer automated with EZ Experiment. Data was collected and exported to Microsoft Excel spreadsheets for analysis.

Culture Growth and Metabolite Analysis

Shake flask experiments were performed using M9 minimal media (Sigma-Aldrich) containing 6.78 g/L disodium phosphate, 3 g/L monopotassium phosphate, 0.5 g/L NaCl, 1 g/L NH 4 Cl, 2 mM MgSO 4 , 100 μM CaCl 2 ), and 40 μM FeSO 4 .7H 2 O supplemented with 20 mM glucose (Fisher Scientific), ethylene glycol, or sodium acetate (Sigma-Aldrich). Cultures were grown in 225 mL baffled shake flasks, incubated at 30° C., 225 rpm. Culture growth was followed by periodic measurement of the optical density at 600 nm (OD 600 ) using a Beckman DU640 spectrophotometer (Beckman Coulter, Brea Calif.). When testing for growth on ethylene glycol, glyxoylate or glycolaldehyde, overnight cultures were spun down, washed in M9 minimal media lacking substrates and resuspended in same media prior to inoculating into shake flasks at OD600=0.1. Growth was conducted in shake flasks under the same conditions described above. Except for experiments conducted with plasmid-bearing strains, all cultures were performed in duplicate. Concentrations of glucose, ethylene glycol, glycolaldehyde, glyoxal, glycolate, glyxoylate, and oxalate in filtered culture supernatant samples were measured by high performance liquid chromatography (HPLC) on an Agilent1100 series system (Agilent USA, Santa Clara, Calif.) utilizing a Phenomenex Rezex RFQ-Fast Fruit H + column (Phenomenex, Torrance, Calif.) and cation H+ guard cartridge (Bio-Rad Laboratories, Hercules, Calif.) at 85° C. A mobile phase of 0.1 N sulfuric acid was used at a flow rate of 1.0 mL/min and a diode array detector was utilized for compound detection. Products were identified by comparing the retention times and spectral profiles with pure compounds.

RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR

To prepare P. putida cultures for RNA extraction, cells were grown up overnight in M9 minimal medium containing 20 mM glucose in baffled shake flasks at 30° C., 225 rpm. Cells were then diluted and transferred to fresh cultures containing 20 mM ethylene glycol and 40 mM sodium acetate @ OD600=0.1, 30° C., 225 rpm. Cells were harvested at mid-exponential growth phase when OD600 reached 0.8-1 by adding cells to 2× volume of Qiagen RNAprotect Bacteria Reagent, mixing well and resting for 5 minutes prior to centrifugation at 5,000×g for 15 min at 4° C. Supernatant was removed and cells were frozen and stored at −80° C. until use. RNA was extracted from cells using Qiagen's RNeasy mini kit following manufacturer's instructions including a DNAse (Qiagen RNase-Free DNase) in column digestion for 1 hour at room temperature following manufacturer's instructions. After isolation, and additional DNase digestion was performed with Ambion's TURBO DNase (Ambion, Austin, Tex., USA) for 2 hours at 37° C. that was followed by an additional RNA purification to remove DNase using the same kit. cDNA was prepared from RNA using an iScript Reverse Transcription supermix kit for RT-qPCR (Bio-Rad). The expression levels of seven genes were analyzed using primers designed by the Realtime PCR tool for RT-qPCR and is listed in Table 4 . Quantitive real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Lab, Hercules, Calif., USA). The reaction conditions were 10 min at 95° C., 39×(15 s at 95° C., 45 s at 55° C., followed by melting curve analysis: 1 min at 95° C., 81×(30 s starting at 55° C., increasing 0.5° C. per cycle, ending at 95° C.). Experiments were performed in triplicate with biological duplicates. The gene expression levels were assessed by the following formula:

Gene expression level=2 Ct(rpoD)−Ct(target)

Ct values represent the first cycle at which the instrument can distinguish the fluorescence of nucleic acid amplification generated as being above the background signal. This method compares the Ct value of the house keeping gene rpoD to the Ct value of the target gene. Final expression levels were averaged for each target gene and normalized to the expression level of the control ( P. putida KT2440) strain. NAD(P)H oxidizing activity assays used for measuring hydroxypyruvate substrate conversion

For extracting whole lysate protein for enzyme assays, strains were grown in LB medium and harvested during exponential growth phase. The cells were collected by centrifugation at 4,800 rpm for 5 min, washed with water twice, and re-suspended in B-PER solution supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, Mass., USA). Whole lysate proteins were obtained by following manufacture recommended protocol (Thermo Fisher Scientific, Waltham, Mass., USA). The protein concentration of samples was assessed using a NanoDrop 2000/c Spectrophotometers (Thermo Fisher Scientific, Waltham, Mass., USA) by following manufacturer protocol. Hydroxypyruvate reduction activity of samples was measured by monitoring oxidation of NAD(P)H at 340 nm with FLUOstar Omega micro plate reader (BMG Labtech, Ortenberg, Germany). Two hundred microliters of a reaction mixture-containing 150 μL of 50 mM potassium phosphate buffer (pH 7), 20 μL of 0.5 M lithium β-hydroxypyruvate hydrate (Sigma-Aldrich, St. Louis, Mo., USA), 20 μL of 0.7 mM NAD(P)H, and 10 μL of the cell extracts were used for the enzyme activity assay. One unit (1 U) of enzyme was defined by the conversion of 1 μM of NAD(P)H into NAD(P) + per minute. The units were normalized to the total protein (mg).

Where nucleotides 1511 to 3286 of SEQ ID NO: 5 are gcl that encodes for glyoxylate carboligase PP4297.

Where nucleotides 3320 to 4213 of SEQ ID NO: 5 are glxR that encode for 2-Hydroxy-3 oxopropionate-reductase PP4299.

Hydroxy-3-oxopropionate-reductase PP4299 has an

amino acid sequence of SEQ ID NO: 7 as follows:

M A K I G F I G T G I M G K P M A Q N L Q K

A G H S L F I S T H H D A A P A D L I A A G

A V A L A N P K E V A Q E A E F I I V M V P

D T P Q V E S V L F G E N G V A E G V G P N

K V V I D M S S I S P T A T K A F A E K I K

A T G A A Y L D A P V S G G E V G A K A A T

L S I M V G G C P N A F E R T L P L F E A M

G K N I T R V G G N G D G Q T A K V A N Q I

I V A L N I Q A V A E A L L F A A K N G A D

P A K V R E A L M G G F A S S K I L E V H A

E R M I K G T F D P G F R I N L H Q K D L N

L A L Q G A K E L G I N L P N T S N A Q Q V

F N T C Q A L G G G N W D H S A L I K G L E

In an embodiment, and as used herein, homologous nucleic acid sequences are about 60%, 65%, 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or even higher identical to nucleic acids disclosed herein. In an embodiment, and as used herein, homologous amino acid sequences are about 60%, 65%, 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95% or even higher identical to amino acids and proteins disclosed herein.

The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.