A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker

Jingqi Chen,Yuanming Gai,Zhaoxia Jin,Dawei Zhang,Jibin Sun

doi:10.1186/s40643-017-0139-7

Abstract

BackgroundFood-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis.ResultsIn this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation.ConclusionThe alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis.

Highlights

Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized
Construction of the food‐grade host strain with deficiency of dal In order to obtain the mutant strain with deficiency of dal, we attempted to knock out the gene dal from B. subtilis chromosome using Cre/lox system
The two flanking fragments upstream and downstream of dal and the fragment lox71-zeo-lox66 cassette were fused into a long DNA fragment by SOE-Polymerase chain reaction (PCR), and the fused fragment was directly transformed into B. subtilis 1A751

Summary

Introduction

Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of d-fructose and is rarely found in nature (Mu et al 2012) It has 70% relative sweetness but 0.3% energy of sucrose and is suggested as an ideal sucrose substitute for food products (Matsuo et al 2002; Oshima et al 2006). It shows important physiological functions, such as blood glucose suppressive effect (Hayashi et al 2010; Iida et al 2008), reactive oxygen species scavenging activity (Matsuo et al 2003), and neuroprotective effect (Takata et al 2005). Food-grade expression systems have been widely developed and investigated for

Methods

Results

Conclusion