Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism.
Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore, Singapore.
Liwei
L
Chen
Jianhua
J
Zhang
Jaslyn
J
Lee
Wei Ning
WN
Chen
D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000096:biosynthesis*; D017353:Gene Deletion; D060847:Metabolic Engineering; D051379:Mice; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D000214:Acyl Coenzyme A; D005230:Fatty Acids, Nonesterified; D029701:Saccharomyces cerevisiae Proteins; D013869:Thiolester Hydrolases; D003066:Coenzyme A Ligases; C511244:Faa1 protein, S cerevisiae; C090206:FAA4 protein, S cerevisiae