Genetic and physiological studies on the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from recombinant Escherichia coli were carried out. A recombinant E. coli strain was constructed to produce the copolymer P(3HB-co-3HV). This was achieved by transforming an E. coli fadR atoC mutant with a multi-copy plasmid containing the PHA biosynthetic genes from Alcaligenes eutrophus.
Vectors containing the PHA biosynthetic genes were constructed, and their stability was investigated. When cells harboring pSYL101 and pSYL102 were cultured to accumulate PHB, a high instability was observed. Two vectors, pSYL103 and pSYL104, were then developed by cloning the parB locus of the plasmid R1. They showed 100% stability even during PHB synthesis over 110 generations.
Some physiological relationships between the formation of P(3HB-co-3HV) and primary metabolic intermediates in E. coli were observed. Accumulation of an acidic by-product, acetic acid, decreased the flux of the acetyl-CoA into the PHA biosynthetic pathway, and had detrimental effect on cell growth. Therefore, reducing acetic acid accumulation was important for efficient P(3HB-co-3HV) synthesis. That was accomplished by the increase of initial acetic acid concentration in the medium and the decrease of growth temperature, resulting in the increment of copolymer production.
The effect of glucose and valeric acid concentration on P(3HB-co-3HV) synthesis was investigated. It was proved that the optimum glucose and valeric acid concentration were 5 g/l and 20 mM, respectively. It was found that catabolite repression was in part responsible for the inefficient copolymer production. To relieve this catabolite repression, addition of various nutritional components was tested. It was shown that the addition of oleic acid resulted in a threefold increase in the level of P(3HV) formation than that obtained without it.
Peptone was found to be the best nitrogen source for the P(3HB-co-3HV) synthesis. The addition of some amino acids reduced the metabolic burden imposed on recombinant E. coli, resulting in the increase of P(3HB-co-3HV) production. Proline showed the beneficial effect on P(3HB-co-3HV) accumulation. The addition of valine and isoleucine, which can be broken down via propionyl-CoA, resulted in the high P(3HV) fraction of the copolymer.
Mass production of P(3HB-co-3HV) was carried out by fed-batch culture with glucose concentration control. Final PHA concentration of 22.5 g/l containing 4.0% 3HV monomer was obtained at 21h. The maximum productivity was 1.07 g $l^{-1}h^{-1}$.
재조합 Escherichia coli로부터의 poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] 생산에 관한연구가 수행되었다. 균주는 Alcaligenes eutrophus로부터 cloning된 poly-(3-hydroxybutyrate) [PHB] biosynthetic gene을 삽입한 E. coli fadR atoC mutant strain을 사용하였다.
먼저 E. coli에서 PHB biosynthetic enzyme을 발현시키기 위한 vector를 제조하였고 stability test를 실시하였다. 그 결과 PHB 축적량이 증가함에 따라 segragational instability가 문제점으로 대두되었다. 이러한 instability 문제를 해결하기 위해 plasmid R1의 parB gene을 삽입한 stable vector를 제조한 결과 100%의 stability를 얻을 수 있었다.
P(3HB-co-3HV) 생성과 다른 여러 metabolic intermediates과의 physiological relationship에 관한 연구를 수행하였다. 재조합 E. coli에서의 발효과정중의 문제점의 하나인 부산물생성 (특히 acetic acid)이 P(3HB-co-3HV)생성에 역효과를 나타낸다는 것을 밝혔고 acetic acid 생성을 줄이기 위한 방법을 시도하였다. 초기 acetic acid 농도를 높여 가면서 실험을 해본 결과 50mM 일 때 최종 acetic acid 생성이 적었고 아울러 높은 P(3HB-co-3HV) copolymer를 얻을 수 있었다. 또한 성장온도를 낮춘 결과 높은 3-HV content를 얻을 수 있었다. 탄소원인 glucose와 valeric acid의 농도의 영향을 살펴보았고 각각 5 g/l와 20mM일 때 효과적인 P(3HB-co-3HV) 생산을 얻을 수 있었다. 이때 발견된 glucose의 catabolite repression 효과를 줄이기 위해 oleic acid를 첨가한 결과 3-HV content를 증가시킬 수 있었다. 질소원으로는 peptone이 P(3HB-co-3HV) 생산에 있어 가장 유리하게 작용하였고 amino acid 첨가는 재조합 균주의 metabolic burden을 줄여주는 효과가 있는 것으로 밝혀졌다. 그 중에서도 valine과 isoleucine은 3-HV content를 증가시키는 효과가 있었고 이것은 이 amino acid의 degradation 과정에서 P(3HV)의 precursor인 propionyl-CoA을 거치기 때문인것으로 추측되어진다.
P(3HB-co-3HV)를 대량생산하기 위해 on-line glucose analyzer를 이용한 유가식 배양을 수행하였다. 그 결과 4.0%의 3-HV를 함유한 22.5g/l의 P(3HB-co-3HV)가 얻어졌고 1.07 g $l^{-1}h^{-1}$의 높은 생산성을 얻을 수 있었다.
