Use of transcriptional profiling & bioinformatics to solve production problems: Eliminating red pigment production in aBacillus subtilisstrain producing hyaluronic acid

Abstract

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.

Tentative biosynthetic pathways of some microbial diketopiperazines

“…10a). In the pulcherrimin biosynthetic pathway, YvmC is the CDPS and mainly produces cLL (Tang et al 2006). CypX encodes a P450, which can oxidate the DKP ring of cLL and convert it to pulcherriminic acid (Cryle et al 2010) (Fig.…”

Microarray Studies in Bacillus subtilis

“…Hayashi et al [86] investigated the effects of perR disruption on global gene expression, while Wei et al [87] analyzed TetL regulon. In an attempt to solve a problem of industrial fermentation where an unwanted red pigment is produced from a recombinant B. subtilis strain synthesizing the heterologous polysaccharide hyaluronic acid (HA), Tang et al [88] compared an apigmented B. subtilis to its parent strain. Among the genes whose transcription was significantly altered in the apigmented mutant, yvmC and cypX were selected as target genes to be deleted based on both bioinformatics and microarray examination.…”

“…Among the genes whose transcription was significantly altered in the apigmented mutant, yvmC and cypX were selected as target genes to be deleted based on both bioinformatics and microarray examination. By disrupting yvmC or cypX genes, Tang et al [88] created strains that did not produce red pigment, and concluded that the combination of transcriptional profiling and bioinformatics is an effective way to improve fermentation processes.…”

Snf2 controls pulcherriminic acid biosynthesis and connects pigmentation and antifungal activity of the yeastMetschnikowia pulcherrima

“…Pulcherrimin was first described in yeasts more than 60 years ago, but only very recently the four genes involved in its biosynthesis and transport in Kluyveromyces lactis have been identified (26), and the structure of the molecule synthesized by yeasts has not been determined by modern analytical methods. However, pulcherrimin is also produced by certain bacteria such as Bacillus licheniformis, where the two genes yvmC and cypX, coding for a cyclodipeptide synthase and cytochrome P450 oxidase, respectively, are responsible for its biosynthesis (27)(28)(29). Expression of yvmC and cypX and the formation of pulcherrimin are intricately regulated by factors such as growth stage, iron availability, or environmental stress (30,31).…”