This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Madsen, S. M. Articles by Israelsen, H. Search for Related Content PubMed PubMed Citation Articles by Madsen, S. M. Articles by Israelsen, H. Agricola Articles by Madsen, S. M. Articles by Israelsen, H.

Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from Staphylococcus carnosus and Characterization of the Enzyme

Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm,¹ Department of Perception & Functionality, Biotechnological Institute, Holbergsvej 10, DK-6000 Kolding, Denmark²

Received 19 February 2002/ Accepted 29 May 2002

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 -keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

This article has been cited by other articles:

• Valsesia, G., Medaglia, G., Held, M., Minas, W., Panke, S. (2007). Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin. Appl. Environ. Microbiol. 73: 1635-1645 [Abstract] [Full Text]  
• Ganesan, B., Dobrowolski, P., Weimer, B. C. (2006). Identification of the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of Lactococcus lactis.. Appl. Environ. Microbiol. 72: 4264-4273 [Abstract] [Full Text]  
• Cernat Bondar, D., Beckerich, J.-M., Bonnarme, P. (2005). Involvement of a Branched-Chain Aminotransferase in Production of Volatile Sulfur Compounds in Yarrowia lipolytica. Appl. Environ. Microbiol. 71: 4585-4591 [Abstract] [Full Text]