Heterologous production of 10-methylstearic acid
A methyl and methylene technology, applied in the production of fatty acids, production of fat, chemical modification of fatty acids, etc., can solve problems such as oxidative degradation
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Embodiment 1
[0189] Example 1: Identification of 10-methylstearic acid genes tmsA, tmsB and tmsC
[0190] Two different genes have been identified to be responsible for 10-methylstearate production in M. tuberculosis (see Meena, L.S. and P.E. Kolattukudy, Biotechnology & Applied Biochemistry 60(4):412 (2013); and Meena, L.S. et al. Biological Chemistry 394(7):871(2013)). Curiously, neither gene was conserved in every 10-methylstearate-producing actinomycete species. Although it is possible that different species of actinomycetes have independently evolved genes for the synthesis of 10-methylstearate, such convergent evolution is rare. A simpler explanation is that a single common gene or gene set is responsible for 10-methylstearate production in Actinomycetes.
[0191] To identify genes likely responsible for 10-methylstearate production in Actinomycetes, genes from multiple species of 10-methylstearate-producing Actinomycetes were combined with genes encoding enzymes that catalyze li...
Embodiment 2
[0200] Example 2: 10-methyl fat in E. coli using tmsB and tmsA genes from different donor organisms acid production
[0201] method:
[0202] Donor bacterial genomic DNA was obtained from the German Collection of Microorganisms (DSMZ, Germany). Plasmids are constructed by standard molecular biology techniques using the "yeast gap repair" method (Shanks, et al., Appl. Microbiol. Biotechnol., 48:232 (1997)). The empty Escherichia coli expression vector pNC53 (SEQ ID NO: 82) was restriction digested with the enzyme PmeI (New England Biolabs, MA), and a double-strand break was generated between the tac promoter and the terminator sequence of trpT on the vector. The tmsAB gene operon was PCR amplified from genomic DNA with primers flanking sequences such that the tmsB ATG start site was integrated into the tac promoter end by homologous recombination. E. coli transcription and translation are driven by the tac promoter. The stop codon of the tmsA gene was similarly integrate...
Embodiment 3
[0208] Example 3: Expression of tmsB and tmsA in Rhodococcus opacus PD630
[0209] The oleaginous bacterium Rhodococcus opacus naturally produces 10-methyl fatty acids at low levels (0.2% of total fatty acids ( et al., Microbiology, 72:5027 (2006)), and additionally have natural homologues of the tmsB and tmsA genes, but the homologues have not been identified in the literature. In this example, the inventors tested whether overexpression of the tmsB and tmsA genes in Rhodococcus opacus could increase 10-methyl branched fatty acid content.
[0210] method:
[0211] Rhodococcus opacus PD630 was obtained from the German Collection of Microorganisms (DSMZ) starting material DSM 44193. Cultures were revived by diluting with 4 mL of LB medium and incubated in a drum drum at 30°C for 3 days. Once apparent growth appeared, 10 μL of broth was added to a single colony on the LB plate and incubated at 30 °C for an additional 3 days. One colony was isolated and named strain NS1104...
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