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정휘민,임대균,김민선,정규열,오민규 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1
Most of the bacteria have been evolved to maximize growth rate with fast consumption of carbon sources from surroundings. However, fast growing phenotype featured overproduction of acetate. There exist several glucose uptake pathways in E. coli. As the glucose transporters were mutated, the growth rate and glucose uptake decreased but acetate overflow was relieved and improved biomass was achieved. Transcriptome analysis was implemented to investigate the change between wild type and mutant systematically. Various transcriptional and metabolic transition were identified in mutant including central metabolism, TCA cycle, alternative transporters, quorum sensing, chemotaxis, flagella synthesis and stress induced response. We attempted to construct robust host strain by introducing several value-added compound (EGFP, γ-aminobutyrate, lycopene) pathway to sugar transporter mutants. Eventually, the specific production yields of above compounds were improved in mutants.
Inhibition of bacterial respiration for enhancing production of reductive compounds in E. coli
정휘민,오민규 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0
Most of the valuable compounds like biofuels are highly reduced state that NAD(P)H should be required for their synthesis. In aerobic condition, cellular reducing equivalent are competitively consumed between reduced-chemical production and respiration. We tried to knock-down the activity of electron transfer chain to increase cellular NADH pools. Bacterial respiration complex are composed of NADH dehydrogenase, Quinone(ol), cytochrome oxidase and ATP synthase. We made combinatorial mutations in two types of NADH dehydrogenase and cytochrome oxidase respectively. The mutant strains exhibited slightly growth defection and increase of acetate production. Although the fitness of mutants were slightly deteriorated, intracellular NADH pools was higly improved. When NADH consuming pathway (i.e. 2,3-butanediol and isobutanol pathway) was introduced to the mutants, their production were enhanced. Furthermore the mutants maintained high NADH/NAD+ ratio independent of variation in aeration.
정휘민,오민규 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0
Lycopene is red colored carotenoid found in red fruits and vegetables. We developed a lycopene producing E. coli through MEP pathway. Firstly, we introduced isoprenoid synthesis enzyme such as Dxs, IspA, Idi and CrtEBI in wild type E. coli. One of the important features of lycopene is that it is accumulated to the cellular membrane because lycopene is highly hydrophobic. Therefore, high cell density culture can be helpful for production of lycopene. We found out that sugar (glucose) transporter mutant showed increased biomass yield and lowered acetate overflow in microorganism when glucose is used as sole carbon source. Therefore we constructed main glucose uptake pathway in E. coli. As the uptake rate of glucose decreases, maximum growth and yield is inhanced though growth rate was deteriorated. When the transcription level of several genes of parental strain and mutants were compared, TCA cycle, respiration and pentose phosphate pathway were up-regulated in sugar transporter mutants.
이정헌,정휘민,정무영,오민규 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.1
We have previously engineered a Klebsiella pneumoniae strain to increase the 1,3-production (1,3-PDO) yield from glycerol. Here, we describe the further engineering of this strain to improve the biomass formation, resulting in an increase in the 1,3-PDO production. The amino acid lysine at the 167th position in citrate synthase was substituted with alanine using genome editing method to reduce the binding affinity of the enzyme to nicotinamide adenine dinucleotide (NADH). In addition, the arcA gene was deleted that resulted in the inhibition of the expression of citric acid cycle genes under limited aeration conditions. As a consequence, the biomass production was enhanced by 34% and 1,3-PDO formation was elevated from 9.58 to 16.71 g/L. The production of 1,3-PDO per dry cell weight enhanced by 30% from 2.40 to 3.11 g·L−1·DCW−1. The phenotypic changes in the strains were confirmed through the analyses of redox ratio, ATP levels, and changes in the expression of genes related to citric acid cycle and 1,3- PDO pathway.