혐기적 포도당 대사 억제 대장균 돌연변이 균주(glf)개발 및 재조합 단백질 생성 숙주로서의 적합성 조사 = Development of E. coli mutant strain(glf) deficient in anaerobic glucose metabolism and its feasibility test for host strain in recombinant protein production
저자
발행사항
서울 : 성균관대학교 대학원, 1995
학위논문사항
학위논문(석사)-- 성균관대학교 대학원: 생물학과 생물학 전공 1996. 2
발행연도
1995
작성언어
한국어
주제어
KDC
475 판사항(4)
발행국(도시)
서울
형태사항
ⅴ, 71p. : 삽도 ; 26cm.
일반주기명
참고문헌 수록
DOI식별코드
소장기관
In. order to produce useful recombinant proteins, E. coli is widely used as a host strain for high cell density aerobic fermentation. Substantial amount of glucose is added into the growth medium for th-e high-density growth of E. coli as well as for the production of recombinant protein by E. coli strain, since this is an inexpensive and readily utilizable carbon and energy source. The major problem in the high-cell density aerobic fermentation is production of fermentative acidic by-products of which acetate is the most predominant. Production of the acidic by-products, especially acetate, is a major factor in the limitation of high-cell density growth and thereby the production of recombinant protein.
In this study, it was tried to develop E. coli host strain enhancing recombinant protein production by constructing strains producing acidic by-products a t reduced levels during aerobic growth in glucose media. The organic acids production by E. coli during the aerobic growth is caused by anaerobic metabolism due to insufficient oxygen supply into the cell. The formation of the acids may be caused by an imbalance between glucose metabolism and respiration. This is a condition in which the influx of carbon into the cell exceeding demands for biosynthesis and the presence of excess NADH_(2) are likely to be involved. It was, therefore, approached in the following two ways for the research objective of developing host strain for recombinant protein production through constructing mutant strain producing organic acids at
reduced level during the aerobic growth in glucose media
First, glf mutant, isolated by this investigator as a mutant defective in anaerobic growth, was characterized especially for its ability of acid production aerobically and was tested for its feasibility as a host strain for recombinant protein production.
Second, single and multiple mutant strains defective in fermentative pathway were constructed in a isogenic wild type strain. They were tested for the ability of acid production and for the feasibility of host strain for recombinant protein production.
glf strain, JL1506, showed reduced degree of growth inhibition by acid production during the aerobic growth in glucose medium. It produced about 2-fold higher cell yield than wild type control, MG1655, after aerobic growth in LB supplemented with 5% glucose. This strain maintained plasmid pMKT2-1 stably enough that 100% of the plasmid were maintained after 18 generations. It produced B -galactosidase and 0-lactamase from the plasmid at levels of 5-fold and 1-fold of MG1655 in specific activities, therefore, the total activities were increased to 10-fold and 2-fold, respectively. This mutant also produced about 4-fold higher amount of lipocortin than MG1655 from plasmid pHT2 carrying human placental cDNA lipocortin gene. Since the strain JL1506 showed promising results in the growth, plasmid stability, and recombinant gene expression both in homologous and heterologous gene like human lipocortin gene, it is very likely that this strain can be developed as a promising host strain not only for a specific but for general recombinant protein production. In order to understand the physiological basis of the mutation effect, the glf- was mapped and found to be locate in E. coli chromosome at 52 min. region where various genes involved in PTS sugar transport. This result suggests that reduced ability of glucose uptake in the mutant can be an explanation of the physiological basis of the mutational effect.
SH6012 was an multiple mutant strain genetically blocked in biosynthetic pathways of the acidic by-products. This multiple mutant were constructed by introducing mutation in pfl(pyruvate formate-lyase >, frd(fumarate reductase >, Idh(lactate dehydrogenase >, pta(phosphotransacetylase >, ack(acetate kinase >, and aria(regulatory gene of aldehyde dehydrogenase and alcohol dehydrogenase production > gene in an isogenic wild type strain MG1655. This sextuple mutant, SH6012, although production of all of the fermentative products are supposed to be blocked genetically, still produced acetate(12.49 mM >, formate(21.17mM >, and lactate(0.21 mM > in the same growth condition. Besides the fermentative products, the strain SH6012 also produced large amount of pyruvate(18.6 mM >, corresponding to 160-fold of wild type control. Production of large amount of pyruvate and probably NADH_(2) enables this strain to be developed as a hyper-producing strain of pyruvate as it is. This strain also can be developed as lactate or ethanol hyper-producing strain upon the introduction of lactate dehydrogenase or ethanol producing genes in a multi-copy plasmids. To explore this possibility, the sextuple mutant was introduced with pet operon, carrying pyruvate decarboxylase and alcohol dehydrogenase gene of Zymomonas mobilis and found to produce ethanol aerobically in 10% glucose minimal medium at 0.3M, corresponding to 19.9-fold or 3.43-fold of wild type without or with the pet operon, respectively.
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