Beta Hydroxylation of Glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-Dependent β-Hydroxylase

ABSTRACTFlocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced byPseudozyma flocculosa, a biocontrol agent, andUstilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of theP. flocculosa fhd1gene and its homolog fromU. maydis,uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted theuhd1gene inU. maydisand analyzed the secreted UA.uhd1deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function ofP. flocculosaFhd1, the corresponding gene was used to complementU. maydisΔuhd1mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated aP. flocculosahomolog of theU. maydislong-chain fatty-acid alpha hydroxylase Ahd1. TheP. flocculosa ahd1gene, which does not reside in the flocculosin gene cluster, was introduced intoU. maydisΔahd1mutant strains.P. flocculosaAhd1 neither complemented theU. maydisΔahd1phenotype nor resulted in the production of β-hydroxylated UA. This suggests thatP. flocculosaAhd1 is not involved in flocculosin hydroxylation.

Identification of Genes Potentially Involved in the Biocontrol Activity of Pseudozyma flocculosa

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100×) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.