Biosynthesis Of Alkanes And Fatty Alcohols In E. Coli Through Moduler Design

Compared with short chain alcohols, alkanes and fatty alcohols with middle tolong chain (C8-C18) are the ideal biofuel molecules as they are structurally andchemically identical to the fossil fuels they seek to replace. In order to increase theoutput of artificial alkane and fatty alcohols, this paper constructed and optimized thebiosynthesis of free fatty acids (FFAs) and alkane/fatty alcohols derived from fattyacyl-acyl carrier protein (ACP) through moduler design. We also constructed a newoxidative pathway for microbial synthesis of middle-chain alkane/fatty alcoholsderived from FFAs, which could synthesized ‘customized’ petroleum-replica fuelmolecules.An alkane biosynthesis pathway derived from fatty acyl-ACP was constructed inE. coli BL21(DE3) chasis cell utilizing the acyl-ACP reductase (AAR) and aldehydedecarbonylase (ADC) as the alkane synthesis moduler.1-pentadecane,8-heptadeceneand trans-9-hexadecen-1-ol were obtained. In order to improve the output of thesystem,18alkane synthesis moduler were constructed. Through the adaptation ofalkane synthesis moduler with chasis cell, as well as fine tuning the expressionalproportion of aar&adc gene, the output of alkanes was improved from3.3mg/L to50.8mg/L. Through comparison of the difference of the transcriptional and proteinexpression level, the bottleneck of the alkane/fatty alcohols biosynthesis derived fromfatty acyl-ACP was proved as the decarboxylation reaction. This bottleneck could bebroken by the fine tuning and adaptation of the expressional proportion of aar&adcgene. Lastly, E. coli K-12and BL21with endA and trxB gene being knocked out,respectively, was proved to be the most suitable chasis cell for alkane biosynthesisderived from fatty acyl-ACP through adaptation of different E. coli chasis cell withRFS10alkane biosynthesis moduler.On the other hand, the FFAs outputs in E. coli BL21(DE3) ΔfadE chasis cell wasenhanced from0.4mg/L to242.2mg/L through inhibition of FFAs β oxidationpathway and optimizing the expression strength of fatty acyl-ACP hydrolysis moduler.The chain length of the FFAs products was controlled from C12to C16. Moreover, α-dioxygenase (α-Dox) from O. sativa and ADC were added on the basis of FFAs (Cn)over-producing chasis cell, and consequently a middle-chain alkane/fatty alcoholsbiosynthesis system with two carbon atom being taken off (Cn-2) from FFAs wasconstructed firstly in the world. Undecanol, tridecanal, tetradecane,1-tridecanol and 1-pentadecanol were detected as the fermentation products. This new alkanes artificialbiosynthesis system could synthesize ‘customized’ saturated alkane/fatty alcoholswith middle chain length (C11-C15), and overcame the deficiency of existing alkaneartificial biosynthesis which cannot control the carbon chain of alkane/fatty alcohols,or the fatty aldehyde synthesis reaction in which demanding ATP and NAD(P)Hprovided from cell. The new artificial middle-chain alkane/fatty alcohols biosynthesissystem also filled the gap of the existing microbial alkane synthesis system whichonly one decarboxylation reaction carried out. This system provides new insight intodeveloping novel means for artificial synthesis of petroleum-replica fuel molecules inmicrobes.