Biosynthesis of C9 to C12 ω-Aminocarboxylic Acids from Renewable Fatty Acids by Using Recombinant Escherichia coli-based Biocatalysts

Abstract

C9 to C12 ω-aminocarboxylic acids are widely used for manufacturing aliphatic polyamide materials. Thereby, this study has investigated multi-step biosynthesis of C9 to C12 ω-aminocarboxylic acids from renewable long chain fatty acids (e.g., oleic acid) via recombinant Escherichia coli-based biocatalysis. First, ω-hydroxynonanoic acid, which had been produced from oleic acid via hydration of fatty acids, oxidation of the hydroxyl group to ketone, Baeyer-Villiger (BVMO) oxidation to ester, and hydrolysis of the ester bond was converted into ω-aminononanoic acid by an alcohol dehydrogenase (ChnD) of Acinetobacter sp. and ω-transaminase of Silicibacter pomeroyi or Agrobacterium fabrum. When the ester bond was hydrolyzed into ω-hydroxynonanoic acid and nonanoic acid, the nonanoic acid showed enzyme inhibition for the subsequent reaction. The microporous polymeric hydrophobic resin of styrene-divinylbenzene (i.e., Sepabeads^TM SP850) was added to remove nonanoic acid. As a result, the ω-aminocarboxylic acid was produced with a conversion of 70% from 100 mM ω-hydroxycarboxylic acid in the presence of 100 mM nonanoic acid using the resin. This biotransformation was then designed to regenerate cofactor of alcohol dehydrogenase. The NADH oxidase (NOX) from Lactobacillus brevis was used to oxidize NADH, which is generated by ChnD. Although the initial bioconversion rate of the recombinant E. coli expressing NOX, ChnD, and ω-transaminase was low, it produced more amine products at high concentrations of substrate than the recombinant E. coli without NOX. The recombinant E. coli biocatalysts expressing the ChnD and ω-transaminase also showed activity to other chain length of ω-hydroxycarboxylic acid. Among C9 to C12 ω-hydroxycarboxylic acids, ω-hydroxydecanoic acid showed the highest initial bioconversion rate which is about 50 U/g dry cells. This study will contribute to biocatalyst engineering for the production of industrially relevant ω-aminocarboxylic acids from renewable fatty acids. And this multi-step biosynthesis could be a new alternative to chemical synthesis.;C9에서 C12 길이의 ω-aminocarboxylic acids는 aliphatic polyamide 물질을 제조하는데 널리 사용된다. 따라서 본 연구는 재조합 대장균 기반 biocatalyst를 이용하여 renewable fatty acid (올레산)로부터 ω-aminocarboxylic acids를 생합성하는 multi-step biosynthesis 경로를 디자인하였다. 먼저, oleatae hydratase (OhyA), Baeyer-Villiger monooxygenase (BVMO), secondary alcohol dehydrogenase (SADH)를 발현시킨 대장균으로 9-(nonanoyloxy)-nonanoic acid (4)를 생성하였고 [1, 2], Thermomyces lanuginosus 유래의 lipase에 의해 nonanoic acid (5)와 ω-hydroxynonanoic acid (6)로 가수분해되었다. 이후 ω-hydroxynonanoic acid (6)에서 ω-aminononanoic acid (8)로 생합성 하는 경로를 디자인하기 위해 Acinetobacter sp. 유래의 alcohol dehydrogenase (ChnD)와 Silicibacter pomeroyi (3HMU) 혹은 Agrobacterium fabrum (Atu3300) 유래의 ω-transaminase가 들어있는 plasmid를 구축하였다. 9-(Nonanoyloxy)nonanoic acid (4)가 가수분해되면서 생성된 nonanoic acid (5)는 이후의 반응에 저해 효과를 나타냈다 [18]. 이러한 저해 효과를 없애기 위해 microporous polymeric hydrophobic resin (SP850)을 사용하여 반응액 중의 nonanoic acid (5)의 농도를 10 mM 이하로 유지하였다. 결과적으로 수지를 사용하여 100 mM ω-hydroxydecanoicc acid 반응을 진행하였을 때 약 60%의 생물 전환 수율로 ω-aminodecanoic acid를 생성하였다. ChnD의 경우 NAD+ 조효소를 반응 중에 필요로 하므로 Lactobacillus brevis 유래의 NADH oxidase (NOX)를 동시발현하여 cofactor regeneration 문제를 해결하였다. NOX와 ChnD, Atu3300을 동시발현 시키는 경우 NOX를 동시발현하지 않았을 때보다 ChnD와 Atu3300의 발현 수준이 낮아져 초기 생물 전환 속도는 감소하였다. 하지만 50 mM ω-hydroxydecanoic acid의 실험 결과, ω-aminodecanoic acid 로의 최종 생물 전환 수율은 더 높았다. 구축한 ChnD, Atu3300 균주의 경우 C9 이외의 길이의 지방산에도 높은 활성을 보였다. C9부터 C12의 ω-hydroxyarboxylic acids 중에서 ω-hydroxydecanoic acid는 50 U/g dry cells로 가장 높은 초기 생물 전환 속도를 나타냈다. 이러한 medium hydroxyl fatty acids 연구는 고온, 고압의 조건을 요하는 화학 합성의 대안이 될 수 있고, ω-aminocarboxylic acids를 합성하는 다양한 산업 공정을 개발하는 연구에 기여할 것이다.