Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase

Abstract

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions.

Engineering the fatty acid synthesis pathway in Synechococcus elongatus PCC 7942 improves omega-3 fatty acid production

“…In Ss7002, Kuo and Khosla determined that FabH was the rate-limiting enzyme of FAS using in vitro reconstituted systems and suggested that, either overexpressing the endogenous fabH gene or by replacement with its E. coli ortholog, the FA flux should be increased [ 17 ]. Attempts to delete fabH in Ss7002 proved impossible, rendering always non-pure deficient mutants and thus suggesting that fabH is essential in this cyanobacterium [ 27 ].…”

“…In Se7942, the fabH deletion was lethal, when assayed by random barcode transposon site sequencing (RB-TnSeq) [ 29 ]. Neither was this gene removed from all chromosomal copies of Ss7002 [ 27 ]. Our attempts to delete fabH in Se7942 also resulted in merodiploid mutants, an indication of the indispensability of this gene, and that no other Se7942 KAS enzyme (e.g., FabF) can act as a functional replacement for FabH.…”

“…Notably, C12:0 is not naturally present in Se7942. Increased levels of MCFAs were reported in an engineered Ss7002 strain in which fabH was replaced by Chaetoceros GSL56 KASIII gene [ 27 ]. MCFAs are appreciated in the fuel industry, which makes fabH overexpression in cyanobacteria potentially useful.…”

Fatty Acids from Microalgae: Targeting the Accumulation of Triacylglycerides

“…GSL56 helped to identify putative enzymes of the FA synthesis pathway. In addition, replacement of ketoacyl-ACP synthase of Synechococcus 7002 with Chaetoceros ketoacyl-ACP synthase III induced FA synthesis [17]. In line with this, TAG accumulating conditions increased the levels of transcripts for KAS in H. pluvialis [15].…”

Photosynthetic Conversion of Carbon Dioxide to Oleochemicals by Cyanobacteria: Recent Advances and Future Perspectives

“…It is noteworthy that these short or medium chain length specific thioesterases should be expressed in the aas mutant to avoid the reactivation and the elongation of FFAs (Table 1). In addition, the replacement of the native FabH with a Chaetoceros ketoacyl-ACP synthase III in the lauric acid-secreting strain of Syn7002 increased MCFA synthesis up to five-fold (Gu et al, 2016).…”