In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.

A third glucose uptake bypass in Corynebacterium glutamicum ATCC 31833.

In Corynebacterium glutamicum, the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) has long been the only known glucose uptake system, but we recently found suppressor mutants emerging from a PTS-negative strain of C. glutamicum ATCC 31833 on glucose agar plates, and identified two alternative potential glucose uptake systems, the myo-inositol transporters encoded by iolT1 and iolT2. The expression of either gene renders the PTS-negative strain WTΔptsH capable of growing on glucose. In the present study, we found a suppressor strain that still grew on glucose even after the iolT1 and iolT2 genes were both disrupted under the PTS-negative background. Whole-genome sequencing of the suppressor strain SPH1 identified a G-to-T exchange at 134 bp upstream of the bglF gene encoding an EII component of the ß-glucoside-PTS, which is found in limited wild-type strains of C. glutamicum. Introduction of the mutation into strain WTΔptsH allowed the PTS-negative strain to grow on glucose. Reverse transcription-quantitative PCR analysis revealed that the mutation upregulates the bglF gene by approximately 11-fold. Overexpression of bglF under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, and deletion of bglF in strain SPH1 abolished the growth again, proving that bglF is responsible for glucose uptake in the suppressor strain. Simultaneous disruption of three glucokinase genes, glk (Cgl2185, NCgl2105), ppgK (Cgl1910, NCgl1835), and Cgl2647 (NCgl2558), in strain SPH1 resulted in no growth on glucose. Plasmid-mediated expression of any of the three genes in the triple-knockout mutant restored the growth on glucose. These results indicate that C. glutamicum ATCC 31833 has an additional non-PTS glucose uptake route consisting of the bglF-specified EII permease and native glucokinases.