Impact of Branched-Chain Amino Acid Catabolism on Fatty Acid and Alkene Biosynthesis in Micrococcus luteus

被引:20
作者
Surger, Maximilian J. [1 ]
Angelov, Angel [1 ]
Stier, Philipp [1 ]
Uebelacker, Maria [1 ]
Liebl, Wolfgang [1 ]
机构
[1] Tech Univ Munich, Dept Microbiol, Munich, Germany
关键词
Micrococcus luteus; branched fatty acid synthesis; olefins; isovaleryl-CoA; isobutyryl-CoA; 2-methylbutyryl-CoA; BCKD complex; branched amino acid catabolism; ISOVALERYL-COA DEHYDROGENASE; BACILLUS-SUBTILIS; STREPTOMYCES-AVERMITILIS; SARCINA LUTEA; BACTERIA; GENES; HYDROCARBONS; ENZYME; MUTANT;
D O I
10.3389/fmicb.2018.00374
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus, whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain alpha-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus. We also show how this knowledge can be used to engineer the isomeric composition and the chain lengths of the olefins produced by this organism.
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页数:14
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共 31 条
[1]   BIOCHEMISTRY OF LONG-CHAIN NONISOPRENOID HYDROCARBONS .I. CHARACTERIZATION OF HYDROCARBONS OF SARCINA LUTEA AND ISOLATION OF POSSIBLE INTERMEDIATES OF BIOSYNTHESIS [J].
ALBRO, PW ;
DITTMER, JC .
BIOCHEMISTRY, 1969, 8 (01) :394-&
[2]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[3]   Novel Flp pilus biogenesis-dependent natural transformation [J].
Angelov, Angel ;
Bergen, Paul ;
Nadler, Florian ;
Hornburg, Philipp ;
Lichev, Antoni ;
Uebelacker, Maria ;
Pachl, Fiona ;
Kuster, Bernhard ;
Liebl, Wolfgang .
FRONTIERS IN MICROBIOLOGY, 2015, 6
[4]   Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle [J].
Angelov, Angel ;
Li, Haijuan ;
Geissler, Andreas ;
Leis, Benedikt ;
Liebl, Wolfgang .
SYSTEMATIC AND APPLIED MICROBIOLOGY, 2013, 36 (08) :585-592
[5]   Structures of isobutyryl-CoA dehydrogenase and enzyme-product complex - Comparison with isovaleryl- and short-chain acyl-CoA dehydrogenases [J].
Battaile, KP ;
Nguyen, TV ;
Vockley, J ;
Kim, JJP .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (16) :16526-16534
[6]   Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA - Comparison with other acyl-CoA dehydrogenases [J].
Battaile, KP ;
Molin-Case, J ;
Paschke, R ;
Wang, M ;
Bennett, D ;
Vockley, J ;
Kim, JJP .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (14) :12200-12207
[7]   Genes Involved in Long-Chain Alkene Biosynthesis in Micrococcus luteus [J].
Beller, Harry R. ;
Goh, Ee-Been ;
Keasling, Jay D. .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2010, 76 (04) :1212-1223
[8]   β-Lactone Synthetase Found in the Olefin Biosynthesis Pathway [J].
Christenson, James K. ;
Richman, Jack E. ;
Jensen, Matthew R. ;
Neufeld, Jennifer Y. ;
Wilmot, Carrie M. ;
Wackett, Lawrence P. .
BIOCHEMISTRY, 2017, 56 (02) :348-351
[10]   A 2ND BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE GENE-CLUSTER (BKDFGH) FROM STREPTOMYCES-AVERMITILIS - ITS RELATIONSHIP TO AVERMECTIN BIOSYNTHESIS AND THE CONSTRUCTION OF A BKDF MUTANT SUITABLE FOR THE PRODUCTION OF NOVEL ANTIPARASITIC AVERMECTINS [J].
DENOYA, CD ;
FEDECHKO, RW ;
HAFNER, EW ;
MCARTHUR, HAI ;
MORGENSTERN, MR ;
SKINNER, DD ;
STUTZMANENGWALL, K ;
WAX, RG ;
WERNAU, WC .
JOURNAL OF BACTERIOLOGY, 1995, 177 (12) :3504-3511