Hyuk Lee

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered Escherichia coli

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1Ps6-19 and PctCp was developed and cultured in a chemically defined medium containing 20xa0g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6xa0mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.

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

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.

Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose

The Escherichia coli XL1-blue strain was metabolically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] through 2-ketobutyrate, which is generated via citramalate pathway, as a precursor for propionyl-CoA. Two different metabolic pathways were examined for the synthesis of propionyl-CoA from 2-ketobutyrate. The first pathway is composed of the Dickeya dadantii 3937 2-ketobutyrate oxidase or the E. coli pyruvate oxidase mutant (PoxB L253F V380A) for the conversion of 2-ketobutyrate into propionate and the Ralstonia eutropha propionyl-CoA synthetase (PrpE) or the E. coli acetyl-CoA:acetoacetyl-CoA transferase for further conversion of propionate into propionyl-CoA. The second pathway employs pyruvate formate lyase encoded by the E. coli tdcE gene or the Clostridium difficile pflB gene for the direct conversion of 2-ketobutyrate into propionyl-CoA. As the direct conversion of 2-ketobutyrate into propionyl-CoA could not support the efficient production of P(3HB-co-3HV) from glucose, the first metabolic pathway was further examined. When the recombinant E. coli XL1-blue strain equipped with citramalate pathway expressing the E. coli poxB L253F V380A gene and R. eutropha prpE gene together with the R. eutropha PHA biosynthesis genes was cultured in a chemically defined medium containing 20xa0g/L of glucose as a sole carbon source, P(3HB-co-2.3xa0mol% 3HV) was produced up to the polymer content of 61.7xa0wt.%. Moreover, the 3HV monomer fraction in P(3HB-co-3HV) could be increased up to 5.5xa0mol% by additional deletion of the prpC and scpC genes, which are responsible for the metabolism of propionyl-CoA in host strains.

Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.

Propionyl-CoA dependent biosynthesis of 2-hydroxybutyrate containing polyhydroxyalkanoates in metabolically engineered Escherichia coli

We have previously reported in vivo biosynthesis of 2-hydroxyacid containing polyesters including polylactic acid (PLA), poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], and poly(3-hydroxybutyrate-co-2-hydroxybutyrate-co-lactate) [P(3HB-co-2HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct(Cp)) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps6-19)). In this study, we further engineered in vivo PLA biosynthesis system in E. coli to synthesize 2HB-containing PHA, in which propionyl-CoA was used as precursor for 2-ketobutyrate that was converted into 2HB-CoA by the sequential actions of Lactococcus lactis (D)-2-hydroxybutyrate dehydrogenase (PanE) and Pct(Cp) and then 2HB-CoA was polymerized by PhaC1(Ps6-19). The recombinant E. coli XL1-blue expressing the phaC1437 gene, the pct540 gene, and the Ralstonia eutropha prpE gene together with the panE gene could be grown to 0.66 g/L and successfully produced P(70 mol%3HB-co-18 mol%2HB-co-12 mol%LA) up to the PHA content of 66 wt% from 20 g/L of glucose, 2 g/L of 3HB and 1 g/L of sodium propionate. Removal of the prpC gene in the chromosome of E. coli XL1-blue could increase the mole fraction of 2HB in copolymer, but the PHA content was decreased. The metabolic engineering strategy reported here suggests that propionyl-CoA can be successfully used as the precursor to provide PHA synthase with 2HB-CoA for the production of PHAs containing 2HB monomer.

Metabolic Engineering of Ralstonia eutropha for the Production of Polyhydroxyalkanoates From Sucrose

A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes β-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that β-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20u2009g/L of sucrose, a high P(3HB) content of 73.2u2009wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5u2009mol% LA) to a polymer content of 19.5u2009wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5u2009mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52u2009×u200910(5) (Mn ) and 2.19u2009×u200910(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered Escherichia coli

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].

Biosynthesis of poly(2-hydroxyisovalerate-co-lactate) by metabolically engineered Escherichia coli.

Polyhydroxyalkanoates (PHAs) containing 2‐hydroxyacids such as lactate (LA) and 2‐hydroxybutyrate (2HB) have recently been produced by metabolically engineered microorganisms. Here, we further expanded 2‐hydroxyacid monomer spectrum of PHAs by engineering Escherichia coli to produce PHAs containing 2‐hydroxyisovalerate (2HIV). To generate 2HIV in vivo, feedback resistant ilvBNmut genes encoding acetohydroxyacid synthase and ilvCD genes encoding ketol‐acid reductoisomerase and dihydroxyacid dehydratase, respectively, and panE gene encoding d‐2‐hydroxyacid dehydrogenase are overexpressed. Also, pct540 gene encoding evolved propionyl‐CoA transferase and phaC1437 gene encoding evolved PHA synthase are overexpressed along with ilvBNmut, ilvCD, and panE genes in E. coli strain for in vivo synthesis of 2HIV containing PHAs. E. coli strain expressing all of these genes can produce poly(13.2 mol% 2HIV‐co‐7.5 mol% 2HB‐co‐42.5 mol% 3HB‐co‐36.8 mol% LA) when it is cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3‐hydroxybutyrate (3HB). To produce PHA containing only 2HIV and LA monomers, poxB, pflB, adhE and frdB genes encoding enzymes involved in competing pathways for pyruvate are deleted so that cells can generate more 2HIV and LA. When this engineered E. coli strain expressing ilvBNmut, ilvCD, panE, pct540 and phaC1437 genes is cultured in the medium containing 20 g/L of glucose and 2 mM l‐isoleucine, which can inhibit l‐threonine dehydratase responsible for in vivo 2HB generation, poly(20 mol% 2HIV‐co‐80 mol% LA) can be produced to the polymer content of 9.6% w/w. These results suggest that novel PHAs containing 2HIV can be produced by engineering branched‐chain amino acid metabolism.

Lipase-catalyzed enantioselective synthesis of (R,R)-lactide from alkyl lactate to produce PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid).

R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method.

Identification of a novel 11β-HSD1 inhibitor from a high-throughput screen of natural product extracts

Selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential as a treatment for metabolic syndrome including type 2 diabetes mellitus and obesity. To identify 11β-HSD1 inhibitors, we conducted high-throughput screening (HTS) of active natural product extracts from the Korea Chemical Bank, including Tanshinone I, Tanshinone IIA, and flavanone derivatives, and 2- and 3-phenyl-4H-chromen-4-one. Then Tanshinone IIA and its derivatives were targeted for the development of a lead compound according to the HTS results. However, the mechanism for anti-adipogenic effect through 11β-HSD1 enzyme inhibition by Tanshinone IIA is not clear. Tanshinone IIA (2a) concentration-dependently inhibited 11β-HSD1 activity in human and mouse 11β-HSD1 overexpressed cells and 3T3-L1 adipocytes. Tanshinone IIA (2a) also inhibited 11β-HSD1 enzyme activities in murine liver and fats. Furthermore, Tanshinone IIA (2a)-suppressed adipocyte differentiation of cortisone-induced adipogenesis in 3T3-L1 cells was associated with the suppression of the cortisone-induced adipogenesis-specific markers mRNA and protein expression. In 3T3-L1 preadipocytes, Tanshinone IIA (2a)-inhibited cortisone induced reactive oxygen species formation in a concentration-dependent manner. Thus, these results support the therapeutic potential of Tanshinone IIA (2a) as a 11β-HSD1 inhibitor in metabolic syndrome patients.