Engineering Saccharomyces cerevisiae for Efficient Biosynthesis of Fatty Alcohols Based on Enhanced Supply of Free Fatty Acids

Abstract

In recent years, production of fatty acid derivatives has attracted much attention because of their wide range of applications in renewable oleochemicals. Microorganisms such as Saccharomyces cerevisiae provided an ideal cell factory for such chemical synthesis. In this study, an efficient strategy for the synthesis of fatty alcohols based on enhanced supply of free fatty acids (FFAs) was constructed. The FAA1 and FAA4 genes encoding two acyl-CoA synthetases in S. cerevisiae were deleted, resulting in the accumulation of FFAs with carbon chain length from C8 to C18. The coexpression of the carboxylic acid reductase gene (car) from Mycobacterium marinum and the phosphopantetheinyl transferase gene (sfp) from Bacillus subtilis successfully converted the accumulated FFAs into fatty alcohols. The concentration of the total fatty alcohols reached 24.3 mg/L, which is in agreement with that of the accumulated FFAs. To further increase the supply of FFAs, the DGAI encoding the acyl-CoA:diacylglycerol acyltransferase involved in the rate-limiting step of triacylglycerols storage was codeleted with FAA1 and FAA4, and the acyl-CoA thioesterase gene (acot) was expressed together with car and sfp, resulting in an enhanced production of fatty alcohols, the content of which increased to 31.2 mg/L. The results herein demonstrated the efficiency of the engineered pathway for the production of fatty acid derivatives using FFAs as precursors.

An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae

“…The production of 1-octanol confirmed that S. cerevisiae contains suitable endogenous ADHs/ALRs for the reduction of 1-octanal (Fig. 2 ) as shown earlier [ 29 ]. Besides 1-octanol, also small amounts of 1-hexanol (5.5 ± 0.6 mg L −1 after 72 h; data not shown) could be determined.…”

“…It is worthy to mention that even the control strain RPY21, expressing wild-type FAS and overexpressing CAR and Sfp, secreted 1-octanol in small amounts (3.9 ± 0.1 mg L −1 ) (Fig. 3 a), which has also been reported before in a comparable setup [ 29 ]. Possibly, this is the result of the small amounts of octanoic acid produced by wild-type FAS or the mitochondrial type II FAS system in S. cerevisiae [ 53 , 54 , 59 ], especially together with deletion of FAA2 as in strain RPY21 [ 58 ].…”

“…2 ) and must be activated by a phosphopantetheine transferase: in this study, Sfp, which attaches the prosthetic group 4′-phosphopantetheine to the enzyme [ 31 , 32 ]. The aldehydes can be further reduced to primary alcohols by endogenous alcohol dehydrogenases (ADH) or aldehyde reductases (ALR) in yeast [ 29 , 33 ].

Fig.

…”

“…A mutant version of S. cerevisiae FAS (Fas1 R1834K /Fas2) produces octanoyl-CoA which is hydrolyzed by endogenous thioesterases (TE) to the free octanoic acid [ 53 ]. A heterologous carboxylic acid reductase (CAR) from M. marinum then converts the free fatty acid to octanal which is further reduced to 1-octanol by endogenous alcohol dehydrogenases (ADH) and aldehyde reductases (ALR) [ 29 ]. CAR must be activated by the phosphopantetheinyl tranferase Sfp from Bacillus subtilis [ 31 ].…”

“…Fatty acyl-CoA reductases (FAR) and acyl-ACP reductases (AAR) proved to be suitable for the reduction of both thioester species, which they directly reduce to the corresponding alcohol [ 17 , 19 – 26 ] and often do not discriminate between ACP and CoA as carrier [ 27 ]. A free carboxylate is comparably unreactive, but can be efficiently converted to a C n-1 aldehyde by α-dioxygenases (αDOX) under decarboxylative elimination of the terminal carboxyl group [ 28 ] or to a C n aldehyde by carboxylic acid reductases (CAR) [ 29 – 31 ]. The CAR-enzyme family requires a phosphopantetheinylation by a phosphopantetheinyl transferase to be active [ 32 ].…”