Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme.
Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5'-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived alpha-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.
Madsen
Søren M
SM
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. sma@bioteknologisk.dk
Beck
Hans Christian
HC
Ravn
Peter
P
Vrang
Astrid
A
Hansen
Anne Maria
AM
Israelsen
Hans
H
D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D017353:Gene Deletion; D005822:Genetic Vectors; D008461:Meat Products / Q000382:microbiology; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
D000597:Amino Acids, Branched-Chain; D003470:Culture Media; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase