Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation

Abstract

Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.

Keywords: Bacillus; Cocoa fermentation; DegU regulator; Pectate lyase; pel gene regulation.