Characterization of cyanobacterial cells synthesizing 10-methyl stearic acid.

Recently, microalgae have attracted attention as sources of biomass energy. However, fatty acids from the microalgae are mainly unsaturated and show low stability in oxygenated environments, due to oxidation of the double bonds. The branched-chain fatty acid, 10-methyl stearic acid, is synthesized from oleic acid in certain bacteria; the fatty acid is saturated, but melting point is low. Thus, it is stable in the presence of oxygen and is highly fluid. We previously demonstrated that BfaA and BfaB in Mycobacterium chlorophenolicum are involved in the synthesis of 10-methyl stearic acid from oleic acid. In this study, as a consequence of the introduction of bfaA and bfaB into the cyanobacterium, Synechocystis sp. PCC 6803, we succeeded in producing 10-methyl stearic acid, with yields up to 4.1% of the total fatty acid content. The synthesis of 10-methyl stearic acid in Synechocystis cells did not show a significant effect on photosynthetic activity, but the growth of the cells was retarded at 34 °C. We observed that the synthesis of 10-methylene stearic acid, a precursor of 10-methyl stearic acid, had an inhibitory effect on the growth of the transformants, which was mitigated under microoxic conditions. Eventually, the amount of 10-methyl stearic acid present in the sulfoquinovosyl diacylglycerol and phosphatidylglycerol of the transformants was remarkably higher than that in the monogalactosyldiacylglycerol and digalactosyldiacylglycerol. Overall, we successfully synthesized 10-methyl stearic acid in the phototroph, Synechocystis, demonstrating that it is possible to synthesize unique modified fatty acids via photosynthesis that are not naturally produced in photosynthetic organisms.

Construction of a cyanobacterium synthesizing cyclopropane fatty acids.

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1(∆9) synthesis in the desA(-)/desD(-) strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

Production of Lacto-N-biose I Using Crude Extracts of Bifidobacterial Cells.

Lacto-N-biose I (LNB) is supposed to represent the bifidus factor in human milk oligosaccharides, and can be practically produced from sucrose and GlcNAc using four bifidobacterial enzymes, 1,3-ß-galactosyl-N-acetylhexosamine phosphorylase, sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase, recombinantly produced by Escherichia coli. Here the production of LNB by the same enzymatic method without using genetically modified enzymes to consider the use of LNB for a food ingredient was reported. All four enzymes were produced as the intracellular enzymes of Bifidobacterium strains. The mixture of the crude extracts contained all four enzymes, with other enzymes interfering with the LNB production, namely, phosphoglucomutase, fructose 6-phosphate phosphoketolase, and glycogen phosphorylase. The first two interfering enzymes were selectively inactivated by heat treatment at 47 °C for 1 h in the presence of pancreatin, and glycogen phosphorylase was disabled by hydrolyzing its possible acceptor molecules using glucoamylase. Finally, 91 % of GlcNAc was converted into LNB in the 100-mL reaction mixture containing 300 mM GlcNAc.

Expression of Genes for a Flavin Adenine Dinucleotide-Binding Oxidoreductase and a Methyltransferase from Mycobacterium chlorophenolicum Is Necessary for Biosynthesis of 10-Methyl Stearic Acid from Oleic Acid in Escherichia coli.

In living organisms, modified fatty acids are crucial for the functions of the cellular membranes and storage lipids where the fatty acids are esterified. Some bacteria produce a typical methyl-branched fatty acid, i.e., 10-methyl stearic acid (19:0Me10). The biosynthetic pathway of 19:0Me10 in vivo has not been demonstrated clearly yet. It had been speculated that 19:0Me10 is synthesized from oleic acid (18:1Δ9) by S-adenosyl-L-methionine-dependent methyltransfer and NADPH-dependent reduction via a methylenated intermediate, 10-methyelene octadecanoic acid. Although the recombinant methyltransferases UmaA and UfaA1 from Mycobacterium tuberculosis H37Rv synthesize 19:0Me10 from 18:1Δ9 and NADPH in vitro, these methyltransferases do not possess any domains functioning in the redox reaction. These findings may contradict the two-step biosynthetic pathway. We focused on novel S-adenosyl-L-methionine-dependent methyltransferases from Mycobacterium chlorophenolicum that are involved in 19:0Me10 synthesis and selected two candidate proteins, WP_048471942 and WP_048472121, by a comparative genomic analysis. However, the heterologous expression of these candidate genes in Escherichia coli cells did not produce 19:0Me10. We found that one of the candidate genes, WP_048472121, was collocated with another gene, WP_048472120, that encodes a protein containing a domain associated with flavin adenine dinucleotide-binding oxidoreductase activity. The co-expression of these proteins (hereafter called BfaA and BfaB, respectively) led to the biosynthesis of 19:0Me10 in E. coli cells via the methylenated intermediate.