Studies on Three 1‐ O ‐acylglycerol‐3‐P Acyl transferases in Clostridium beijerinckii

Abstract

The biosynthesis of phospholipids in bacteria begins with the acylation of sn-glycerol-3-P. The second step is the acylation of the resulting 1-O-acyl-glycerol-3-P catalyzed by PlsC to form phosphatidic acid. Clostridium beijerinckii has two genes annotated as plsC and a related third gene, plsD, of unknown function. We have used CRISPR-Cas9 to delete each of these genes and examined the effects on phospholipid chain compositions. Deletion of Cbei_5028 (plsC) had no effect on the composition of the acyl or alk-1-enyl chains of the lipids. Deletion of Cbei_1741 (plsC) resulted in polar lipids with fewer unsaturated chains in diacyl lipids and plasmalogens. Deletion of Cbei_0579 (plsD) resulted in longer chains in all the lipids examined. It appears that Cbei_1741 (PlsC) is selective for unsaturated fatty acyl chains and that Cbei_0579 (PlsD) is a 1-O-acyl-sn-glycerol-3-P acyltransferase selective for shorter acyl chains. Strain characterization experiments demonstrated that ∆Cbei_1741 mutant had slightly increased tolerance to butanol compared to the wild type, possibly due to the more saturated chains in diacyl lipids and plasmalogens in the cell membrane. More dramatically, ∆Cbei_1741 mutant grew much faster and produced 40% more butanol (with more glucose consumed) than the wild type in a regular batch fermentation (at 35 oC). Comparatively, both ∆Cbei_5028 and ∆Cbei_0579 mutant strains produced slightly lower butanol than wild type under this condition. Interestingly, ∆Cbei_0579 produced 40% more butanol than the wild type when the fermentation was conducted at 20 oC. While at higher temperature (40 oC), the butanol yield in ∆Cbei_0579 reached 0.32 g/g (33% higher than in wild type) although the final butanol titer was actually slightly lower than in wild type.