The Comparisons of Fatty Acid Composition in Some Anaerobic Gut Fungi Neocallimastix, Orpinomyces, Piromyces, and Caecomyces.

The objective of this study were to identify the fatty acid composition for decanoic (C10:0), tridecanoic (C13:0), myristic (C14:0), pentadecanoic (C15:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1n9c), linoleic (C18:2n6c), arachidic (C20:0), arachidonic (C20:4n6), heneicosanoic (C21:0), erucic (C22:1n9) and Cis-4,7,10,13,16,19-docosahexaenoic (C22:6n3) acids by Neocallimastix, Orpinomyces, Caecomyces and Piromyces species of rumen fungus during in vitro culture. Fatty acid (FA) profi le of anaerobic fungi comprises carbon chains of length ranging from 10 to 22 were analyzed as methyl esters. Analysis of fatty acids was performed using Gas Chromatography-Mass Spectrophotometer (GC-MS). FA measures are presented as proportions of relative amounts (% total fatty acid). The highest amounts of fatty acids for all samples were found as myristic (C14:0) acid. The tridecanoic (C13:0) acid represented the second abundant FA in the fungi in all experimental groups. Stearic acid (C18:0) was the third major fatty acid for isolates investigated in the current study. In addition, another fatty acid was palmitic (C16:0) acid with relative amount representing >20 % of total FA in all samples. Pentadecanoic (C15:0) acid could not be found in any other samples except Orpinomyces sp. (GMLF5). It is concluded that biohydrogenation of fatty acid composition by anaerobic gut fungi are very variable.

Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcus thermophilus and Their Effects on Metabolite Formation.

Streptococcus thermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp. bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level.

Molecular study on cloned endoglucanase gene from rumen bacterium.

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 alpha-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.