Young Hoon Oh

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals.

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putidadavAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20g/L of glucose and 10g/L of L-lysine, 3.6g/L of 5AVA was produced by converting 7g/L of L-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced L-lysine synthesis, 0.27 and 0.5g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20g/L glucose, 10g/L L-lysine and 10g/L α-ketoglutarate, 1.7g/L of glutarate was produced.

Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers

Biochemical conversion of biomass into biofuels, biochemicals, and biopolymers has attracted much interest throughout the world in terms of biorefineries. Lignocellulosic biomass is one of the most plentifully available biomass resources on the earth. It is composed of three main biopolymers - cellulose, hemicelluloses, and lignin, all of which are cross-linked to each other to resist degradation by enzymes and microorganisms resulting in so-called biomass recalcitrance. The biorefinery process typically consists of three steps: pretreatment, hydrolysis, and fermentation. Energy and cost efficiency of biorefinery is predominantly dependent on how to produce inexpensive sugars from complex cell wall component of lignocellulosic biomass by overcoming biomass recalcitrance. There have been tremendous efforts to develop effective biomass pretreatment technologies for obtaining the highest yield of fermentable sugars from biomass feedstocks at the lowest cost. The present review discusses various pretreatment technologies to understand how to effectively break down biomass into fermentable sugars that are eventually used for microbial fermentation to produce biomass-based fuels, chemicals, and polymers.

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.

High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis

L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes.

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 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% 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-21.5 mol% LA) to a polymer content of 19.5 wt% 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-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(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.

Development of rice bran treatment process and its use for the synthesis of polyhydroxyalkanoates from rice bran hydrolysate solution

Rice bran treatment process for the production of 43.7 kg of hydrolysate solution containing 24.41 g/L of glucose and small amount of fructose from 5 kg of rice bran was developed and employed to produce polyhydroxyalkanoates in recombinant Escherichia coli and Ralstonia eutropha strains. Recombinant E. coli XL1-Blue expressing R. eutropha phaCAB genes and R. eutropha NCIMB11599 could produce poly(3-hydroxybutyrate) with the polymer contents of 90.1 wt% and 97.2 wt%, respectively, when they were cultured in chemically defined MR medium and chemically defined nitrogen free MR medium containing 10 mL/L of rice bran hydrolysate solution, respectively. Also, recombinant E. coli XL1-Blue and recombinant R. eutropha 437-540, both of which express the Pseudomonas sp. phaC1437 gene and the Clostridium propionicum pct540 gene could produce poly(3-hydroxybutyrate-co-lactate) from rice bran hydrolysate solution. These results suggest that rice bran may be a good renewable resource for the production of biomass-based polymers by recombinant microorganisms.

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)].

Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process

This study examined nine expired industrial Corynebacterium glutamicum strains with high lysine producing capability for enhanced production of 5-AVA. C. glutamicum KCTC 1857 exhibiting the highest lysine production was transformed with either original Pseudomonas putida davBA genes, encoding the 5-AVA biosynthesis pathway, or C. glutamicum codon-optimized davBA genes. C. glutamicum KCTC 1857 expressing the original genes had superior cell viability and 5-AVA production capability compared to the other strain. This strain produced 39.93g/L of 5-AVA, which is the highest titer reported to date in fed-batch fermentation from glucose. Indeed, Miscanthus hydrolysate solution prepared from a novel process, comprising pretreatment, hydrolysis, purification, and concentration, was used as feedstock for 5-AVA production. A total of 12.51g/L 5-AVA was produced from the Miscanthus hydrolysate; this value is 34.7% higher than that obtained from glucose in batch fermentation.

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.