Use of transcriptional profiling & bioinformatics to solve production problems: Eliminating red pigment production in a Bacillus subtilis strain producing hyaluronic acid

The production of pigments during industrial fermentations is undesirable, and expensive purification steps are often required to remove colored compounds from anticipated commercial products. We observed that a recombinant Bacillus subtilis strain synthesizing the heterologous polysaccharide hyaluronic acid (HA) produced copious amounts of a red pigment with biochemical properties characteristic of pulcherrimin, a previously characterized iron-binding pyrazine compound. An apigmented B. subtilis mutant was isolated following chemical mutagenesis and compared to its parent strain using DNA microarray transcriptome analysis. Among the genes whose transcription was significantly (p < 0.05) altered in the apigmented mutant, yvmC, and cypX were selected as likely pulcherrimin biosynthetic genes on the basis of in silico bioinformatics examination which suggested that the yvmC gene product contains a putative phosphopantetheine domain characteristic of some cyclic peptide synthases, and the cypX gene encodes a cytochrome P450-like enzyme. Pulcherrimin biosynthesis was previously proposed to occur via cyclization of two leucine molecules followed by a redox reaction involving molecular oxygen. Additionally, yvmC and cypX genes are juxtaposed on the B. subtilis chromosome and appear to be coordinately expressed based on hierarchical cluster analysis of microarray data. Disruption of the yvmC or cypX genes yielded strains that did not produce red pigment. This study illustrates that the combination of transcriptional profiling and bioinformatics is an effective approach that can be employed to solve industrial fermentation problems.