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One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli

Nature Biotechnology volume 34, pages 435–440 (2016)Cite this article

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Abstract

Poly(lactate-co-glycolate) (PLGA) is a widely used biodegradable and biocompatible synthetic polymer. Here we report one-step fermentative production of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase that polymerizes D-lactyl-CoA and glycolyl-CoA into PLGA. Introduction of the Dahms pathway enables production of glycolate from xylose. Deletion of ptsG enables simultaneous utilization of glucose and xylose. An evolved propionyl-CoA transferase converts D-lactate and glycolate to D-lactyl-CoA and glycolyl-CoA, respectively. Deletion of adhE, frdB, pflB and poxB prevents by-product formation. We also demonstrate modulation of the monomer fractions in PLGA by overexpressing ldhA and deleting dld to increase the proportion of D-lactate or by deleting aceB, glcB, glcD, glcE, glcF and glcG to increase the proportion of glycolate. Incorporation of 2-hydroxybutyrate is prevented by deleting ilvA or feeding strains with L-isoleucine. The utility of our approach for generating diverse forms of PLGA is shown by the production of copolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate or 2-hydroxyisovalerate.

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Figure 1: Metabolic engineering of E. coli for the production of PLGA and its copolymers.
Figure 2: Characterization of PLGA and its copolymers produced by metabolically engineered E. coli.

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Acknowledgements

We thank S. Choi for the pTrc99s4 plasmid. This work was supported by the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation of Korea ((NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) to S.Y.C., W.J.K., J.E.Y., S.J.P. and S.Y.L.).

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Author notes

  1. So Young Choi and Si Jae Park: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea

    So Young Choi, Won Jun Kim, Jung Eun Yang & Sang Yup Lee

  2. Department of Environmental Engineering and Energy, Myongji University, Yongin, Gyeonggido, Republic of Korea.,

    Si Jae Park

  3. Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea

    Hyuk Lee

  4. Green Chemistry and Engineering Division, Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea

    Jihoon Shin

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Contributions

S.Y.L., S.J.P. and S.Y.C. generated ideas and designed research. S.Y.C., S.J.P. and J.E.Y. performed research and analytical experiments. W.J.K. performed in silico metabolic simulation. S.Y.C., S.J.P., S.Y.L., H.L. and J.S. analyzed data. S.Y.C., S.J.P. and S.Y.L. wrote the paper.

Corresponding author

Correspondence to Sang Yup Lee.

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Competing interests

S.Y.L., S.Y.C. and S.J.P. are authors of a patent filed on PLGA (KR10-2016-0010549), and S.Y.L. and S.J.P. are authors of patents filed on PLGA (PCT/KR2012/0017540, EP12757647.8). S.Y.L. and S.J.P. are also authors of related patents registered on lactate-containing polymers and methods (KR10-1575585-0000, US08883463, JP5820363, KR10-1575585-0000, KR10-1211767-0000, KR10-1114912-0000, KR10-1085960-0000, PCT/KR2007/005858, KR10-0957776-0000, KR10-0957773-0000, US08765402, CN200780043134.X, IDP0029625, KR10-0948777-0000, KR10-0957775-0000, KR10-0957774-0000, KR10-0926491-0000, KR10-0926492-0000, KR10-0926488-0000, KR10-0926489-0000, US08383379, KR10-0979694-0000, TWI376417, EP01885857, CNZL200680018501.6, ARP060102144, MY-150715-A, KR10-0957777-0000).

Integrated supplementary information

Supplementary Figure 1 Results of in vitro Pct540 assay.

(a) Schematic representation of reactions used in the assay. (b) SDS-PAGE of the purified his-tagged Pct540. The lanes are: T, total proteins; S, soluble protein fraction; U, unbound protein fraction; W, washed fraction; E, eluted fraction; L, molecular weight marker (kDa). (c) The specific activities toward d-lactate (LA), glycolate (GA), 4-hydroxybutyrate (4HB) and 2-hydroxyisovalerate (2HIV) (μmol min−1 mg−1). Error bars represent the s.d. of experiments conducted in duplicates.

Supplementary Figure 2 Contents and mole fractions of polymers produced by expressing various Pseudomonas sp. MBEL 6-19 phaC mutants in E. coli XL1-Blue.

(a) Polymer contents and compositions. 2 g per liter of sodium 3-hydroxybutyrate and 2 g per liter sodium glycolate were fed into culture medium to directly supply monomers. 3HB, GA, and LA indicate 3-hydroxybutyrate, glycolate, and d-lactate, respectively. (b) 1H NMR spectrum of polymer produced in E. coli XL1-Blue harboring pPs619C1437Pct540 in CDCl3 solvent. The poly(d-lactate-co-glycolate-co-3HB) with the highest fractions of d-lactate (31.6 mol%) and glycolate (17.2 mol%) was produced. The peaks representing GA, LA and 3HB are shown in red, blue, and purple, respectively.

Supplementary Figure 3 The time profiles of cell growth (OD600) and production of d-lactic acid and glycolic acid.

(a) Cell growth (OD600). (b) d-Lactic acid. (c) Glycolic acid. XBC indicates E. coli XL1-Blue harboring pTac15k (control strain). XB (E. coli XL1-Blue), XB-p, X15-p, X15l-p, X15ld-p and X17ld-p strains harboring pTacxylBC were cultivated in 100 mM MOPS-based MR medium. 20 g per liter of xylose was added as a sole carbon source for XBC and XB. The XB-p, X15-p, X15l-p, X15ld-p and X17ld-p strains were cultured in the medium containing 10 g per liter each of xylose and glucose. Error bars represent the s.d. of experiments conducted in triplicates.

Supplementary Figure 4 In silico genome-scale analysis of the metabolic changes caused by introducing the Dahms pathway in E. coli.

(a) Metabolic fluxes determined based on the consumed xylose and produced metabolites (ethylene glycol and glycolate). (b) Flux-sum differences of metabolites between a control strain (XL1-Blue) and a Dahms pathway-utilizing strain (XL1-Blue harboring pTacxylBC) using xylose as sole carbon source. (c) Flux-sum differences of metabolites between a control strain and an engineered strain (the Dahms pathway-utilizing strain XL1-Blue harboring pTacxylBC) using xylose and glucose as carbon sources to recover the growth rate. (d) In silico metabolic flux values for the reactions in glycolysis calculated by genome-scale flux balance analysis under the objective function of maximization of growth rate. Lower and upper bounds were calculated by using flux variability analysis under the constraint of 95% maximum growth rate and flux values are shown in the parenthesis. Negative number indicates reverse direction of the reaction.

Supplementary Figure 5 The time profiles of residual carbon sources and produced metabolites.

(a) XB-p strain harboring pTacxylBC consuming glucose and xylose simultaneously. (b) The major metabolites, acetic acid and formic acid, produced in XB-p and X15-p strains. “-f” and “-a” indicate formic acid and acetic acid, respectively. Error bars represent the s.d. of experiments conducted in triplicates.

Supplementary Figure 6 GC-MS and 13C NMR analyses of polymers produced in X15l-p strain harboring pTacxylBC and pPs619C1437Pct540.

(a) The GC-MS profile of methylated monomers prepared by methylation of the polymer produced in X15l-p strain harboring pTacxylBC and pPs619C1437Pct540 as described in Online Methods. (b) 13C NMR spectra of polymer analyzed in CDCl3 solvent. GA, LA, and 2HB indicate glycolate, d-lactate, and 2-hydroxybutyrate, respectively.

Supplementary Figure 7 Fed-batch fermentation of X15ld-p strain harboring pTacxylBC and pPs619C1437Pct540.

(a) Time profiles of cell growth (OD600) and concentrations of two carbon sources (glucose, xylose) and products (d-lactic acid, glycolic acid, acetic acid and ethylene glycol). (b) Polymer contents and compositions. After 72 hours of cultivation, poly(d-lactate-co-glycolate-co-2-hydroxybutyrate) at 65.9 mol% d-lactate, 32.6 mol% glycolate and 1.5 mol% 2-hydroxybutyrate was produced with a polymer content of 40.4 wt% of dry cell weight. (c) The phase-contrast microscopic images of cells at 12 h and 72 h. No polymer granules are observed in 12 h, while polymers are clearly visible as shining granules in 72 h.

Supplementary Figure 8 Fed-batch fermentation of X15ld-p strain harboring pTacxylBC and pPs619C1437Pct540.

To prevent the incorporation of 2-hydroxybutyrate, IlvA was inhibited by adding 5 mM of isoleucine in the culture medium. (a) Time profiles of cell growth (OD600) and concentrations of two carbon sources (glucose, xylose) and products (d-lactic acid, glycolic acid, acetic acid and ethylene glycol). (b) Polymer contents and compositions. After 84 hours of cultivation, poly(d-lactate-co-glycolate) at 70.5 mol% d-lactate and 29.5 mol% glycolate was produced with a polymer content of 36.2 wt%. (c) The phase-contrast microscopic images of cells at 12 h and 72 h. No polymer granules are observed in 12 h, while polymers are clearly visible as shining granules in 72 h.

Supplementary Figure 9 NMR analyses of poly(d-lactate-co-glycolate-co-3-hydroxybutyrate).

(a) 1H and (b) 13C NMR spectra of poly(d-lactate-co-glycolate-co-3-hydroxybutyrate) produced by the X17ld-p strain harboring pTacxylBC_phaAB and pPs619C1437Pct540 in a medium containing xylose, glucose and 5 mM L-isoleucine.

Supplementary Figure 10 NMR analyses of poly(d-lactate-co-glycolate-co-4-hydroxybutyrate).

(a) 1H and (b) 13C NMR spectra of poly(d-lactate-co-glycolate-co-4-hydroxybutyrate) produced by the X17ld-pyg strain harboring pTacxylBC_s4D and pPs619C1437Pct540 in a medium containing xylose, glucose and 5 mM L-isoleucine.

Supplementary Figure 11 NMR analyses of poly(d-lactate-co-glycolate-co-2-hydroxyisovalerate).

(a) 1H and (b) 13C NMR spectra of poly(d-lactate-co-glycolate-co-2-hydroxyisovalerate) produced by the X17ld-p strain harboring pTacxylBC and pPs619C1437Pct540 in a medium containing xylose and glucose and additionally supplemented with 5 mM L-isoleucine and 2 g per liter of 2-hydroxyisovaleric acid.

Supplementary Figure 12 NMR analyses of poly(d-lactate-co-glycolate-co-5-hydroxyvalerate).

(a) 1H and (b) 13C NMR spectra of poly(d-lactate-co-glycolate-co-5-hydroxyvalerate) produced by the X17ld-p strain harboring pTacxylBC and pPs619C1437Pct540 in a medium containing xylose and glucose and additionally supplemented with 5 mM L-isoleucine and 2 g per liter of sodium 5-hydroxyvalerate.

Supplementary Figure 13 NMR analyses of poly(d-lactate-co-glycolate-co-6-hydroxyhexanoate).

(a) 1H and (b) 13C NMR spectra of poly(d-lactate-co-glycolate-co-6-hydroxyhexanoate) produced by the X17ld-p strain harboring pTacxylBC and pPs619C1437Pct540 in a medium containing xylose and glucose and additionally supplemented with 5 mM L-isoleucine and 2 g per liter of 6-hydroxyhexanoic acid.

Supplementary Figure 14 LC-MS analyses of 5-hydroxyvaleryl-CoA and 6-hydroxyhexanoyl-CoA.

(a) 5-hydroxyvaleryl-CoA and (b) 6-hydroxyhexanoyl-CoA generated by Pct540 during the in vitro enzyme assay. The LC-MS samples were prepared by extraction of CoA derivatives from reaction mixture after in vitro Pct540 enzyme assay as described in Online Methods.

Supplementary Figure 15 DSC analyses of PLGA copolymers.

The curves show thermal properties of the copolymer containing 23.5 mol% 3-hydroxybutyrate (3HB), 9.1 mol% 4-hydroxybutyrate (4HB), 6.1 mol% 5-hydroxyvalerate (5HV) 1.6 mol% 6-hydroxyhexanoate (6HHA). The properties of the analyzed copolymer are shown in Table 1 and Supplementary Table 3.

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Supplementary Figures 1–15, Supplementary Tables 1–6 and Supplementary Notes 1–5 (PDF 2120 kb)

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Choi, S., Park, S., Kim, W. et al. One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli. Nat Biotechnol 34, 435–440 (2016). https://doi.org/10.1038/nbt.3485

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