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

Abstract

Background:The biosynthetic pathway for staphyloxanthin has previously been proposed to consist of five enzymes. Results: A sixth pathway enzyme, 4,4Ј-diaponeurosporen-aldehyde dehydrogenase, was identified using a synthetic module approach. Conclusion:The complete staphyloxanthin biosynthetic pathway consists of six enzymes in Staphylococcus aureus. Significance: This is the first report demonstrating the complete staphyloxanthin pathway.The biosynthetic pathway for staphyloxanthin, a C 30 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 Staphylococcus aureus AirSR Two-Component System Mediates Reactive Oxygen Species Resistance via Transcriptional Regulation of Staphyloxanthin Production

“…, that is the product of the biosynthetic pathway encoded by crtOPQMN and aldH (3,4). The initial observational data suggested golden-pigment production was linked to AirSR TCS expression.…”

“…The distinctive golden pigment of S. aureus is the biosynthetic product of crtOPQMN and aldH (3,4). The final product of this six-enzyme-biosynthetic pathway is a membrane-embedded golden C 30 triterpenoid carotenoid, ␣-D-glucopyranosyl-1-O-(4,4=-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate), termed staphyloxanthin (STX) (5).…”

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

“…The carotenoid biosynthetic pathways of Pantoea have been successfully used as basic pathways for many carotenoid studies (11)(12)(13). The pathway enzymes from different sources have occasionally shown unusual activities or substrate affinities in a heterologous host (14)(15)(16). Carotenogenic enzymes, especially carotenoid-modifying enzymes, tend to be promiscuous in their substrate specificity (14) and show unexpected/hidden activities (3,16) when expressed in heterologous host microorganisms.…”

“…The pathway enzymes from different sources have occasionally shown unusual activities or substrate affinities in a heterologous host (14)(15)(16). Carotenogenic enzymes, especially carotenoid-modifying enzymes, tend to be promiscuous in their substrate specificity (14) and show unexpected/hidden activities (3,16) when expressed in heterologous host microorganisms. Therefore, the activities of carotenoidmodifying enzymes (e.g., spheroidene monooxygenase CrtA and CrtZ) or important pathway branch point enzymes (e.g., CrtY) in heterologous hosts have been the focus of study.…”

New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

“…Synthetic biological approaches have been widely used for the redesign and reconstruction of biosynthetic pathways in heterologous hosts (18,19). The functional, modular expression of redesigned pathway enzymes can improve the productivity and yield of target molecules (20).…”