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Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

ABSTRACT: Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. Enzymatic studies, synthetic chemistry, NMR spectroscopy and mass spectrometry approaches suggest that, in contrast to the prevailing consensus, octanoate is not esterified to the primary hydroxyl group of glycerate but instead to the C6 OH of the second glucose in DGG. These observations raise important new questions about the MGLP reducing end architecture and about subsequent biosynthetic steps. Functional characterization of this unique octanoyltransferase, whose gene has been proposed to be essential for M. tuberculosis growth, adds new insights into a vital mycobacterial pathway, which may inspire new drug discovery strategies.

SUBMITTER: Maranha A

PROVIDER: S-EPMC4555173 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

Maranha Ana A Moynihan Patrick J PJ Miranda Vanessa V Correia Lourenço Eva E Nunes-Costa Daniela D Fraga Joana S JS José Barbosa Pereira Pedro P Macedo-Ribeiro Sandra S Ventura M Rita MR Clarke Anthony J AJ Empadinhas Nuno N

Scientific reports 20150901

Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. E ...[more]

PMID: 26324178

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Project description:4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

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Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

Publications

Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

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