Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

• Published in: The Journal of biological chemistry 2012-06, Vol.287 (26), p.21575-21583
• Main Authors: Kim, Se Hyeuk, Lee, Pyung Cheon
• Format: Article
• Language: English
• Subjects: Aldehyde Oxidoreductases - biosynthesis; Aldehyde Oxidoreductases - chemistry; Aldehydes; Alleles; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Biotechnology; Biotechnology - methods; Carotenoid; Carotenoids - metabolism; Catalysis; Chromatography, High Pressure Liquid - methods; Chromatography, Liquid - methods; Diaponeurosporen-aldehyde Dehydrogenase; Escherichia coli - metabolism; Functional Expression; Gene Expression Regulation, Bacterial; Mass Spectrometry - methods; Metabolic Engineering; Microbiology; Models, Biological; Operon; Photosynthesis; Photosynthetic Pigments; Pigmentation; Protein Engineering - methods; Staphylococcus aureus; Staphylococcus aureus - metabolism; Staphyloxanthin; Synthetic Biology; Synthetic Operon; Triterpenes - metabolism; Xanthophylls - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.343020

The biosynthetic pathway for staphyloxanthin, a C30 carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4′-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4′-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4′-diaponeurosporen-4-al into 4,4′-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4′-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes. The biosynthetic pathway for staphyloxanthin has previously been proposed to consist of five enzymes. A sixth pathway enzyme, 4,4′-diaponeurosporen-aldehyde dehydrogenase, was identified using a synthetic module approach. The complete staphyloxanthin biosynthetic pathway consists of six enzymes in Staphylococcus aureus. This is the first report demonstrating the complete staphyloxanthin pathway.