title: Fatty Acyl Incorporation in the Biosynthesis of WAP-8294A, a Group of Potent Anti-MRSA Cyclic Lipodepsipeptides.
abstract: WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster inLysobacter enzymogenesOH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes inE. coliand purified six of the proteins. While these enzymes exhibit a varied level of activityin vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided bothin vivoandin vitroevidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA.
authors.forename: Haotong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Olson
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Su
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA.
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Dussault
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304, USA.
authors.forename: Liangcheng
authors.initials: L
authors.lastname: Du