Construction Of Recombinant Strains Capable Of Producing Alkanes

Considerable consumption of crude oil is severely depleting the abundance of fossil fuels and increases carbon dioxide levels in the atmosphere. Therefore, the investigation on utilizing renewable resource to substitute fossil fuels is becoming an exigent research topic. Alkanes, which are the main composition of traditional fossil fuels, fall short in scientific research. Thus, we have focused on the above topic and successfully cultivated the engineered Escherichia coli that permit alkanes acquisition from endogenous fatty acids within the host bacteria by expression the key enzymes in alkanes biosynthetic pathway, which is a significant milestone marking the practical conversion from renewable biomass to fossil energy.The main results are as follows.(1) Design and verification the feasibility of the alkane biosynthetic pathway in E. coli. Two different genes named acrl and acrM from Acinetobacter encoding acyl-CoA reductase were introduced into engineered E. coli separately. Next, conversion of fatty aldehydes to corresponding alkanes was performed by cloning and expressing the fatty aldehyde decarbonylase, encoded by the gene dc from N. punctiforme strain. The results showed that cells harboring pETDuet-acrM-dc produced tridecane and pentadecane corresponding to each exogenous fatty acids, which provided suitable enzymes for medium-chain alkanes biosynthesis in the engineered E. coli.(2) Genetic control over alkane chain length. The lauric acid (C12:0) and myristic acid (C14:0) became predominant constituents instead of palmitic acid (C16:0) and oleic acid (C18:1) after introducing the esterase ucFatB, which could act as medium-chain substrates in the alkane biosynthetic pathway. The final engineered strain harbouring pETDuet-uc-acrM-dc produced up to 6.43±0.25 mg·g-1 different kinds of medium-chain alkanes, with 2.03±0.07 mg·g-1 undecane,1.51±0.18 mg·g-1 tridecane and 2.89± 0.37 mg·g-1 pentadecane, respectively.(3) Modification to the fatty acyl-CoAs pool to improve alkane production. Since the accumulation of substrates could push the metabolic flux to the final product on certain level, we decided to enhance the formation of the major intermediate fatty acyl-CoAs by overexpression of fadD gene, the endogenous gene from E. coli encoding fatty acyl-CoA synthetase. After coexpression with pACYCDuet-fadD, the production of alkanes raised up to a total amount of 8.05±0.37 mg·g-1 which consisted of 2.21±0.18 mg·g-1 undecane,1.83±0.12 mg·g-1 tridecane and 4.01±0.43 mg·g-1 pentadecane. To improve the production of alkanes, ACP synthase and fatty acid synthase complex from Jatropha curcas was coexpressed together with the alkane biosynthetic pathway, but the results remains to be found in a follow-up study.