Production of d-Tagatose by Whole-Cell Conversion of Recombinant Bacillus subtilis in the Absence of Antibiotics.

Xian Zhang,Zhiming Rao,Meijuan Xu,Qiang Wang,Ruiqi Lu,Taowei Yang,Rongzhen Zhang,Mengkai Hu,Zhiyue Li

doi:10.3390/biology10121343

Abstract

Simple Summaryd-tagatose is a valuable monosaccharide in the food industry produced from lactose by β-galactosidase and arabinose isomerase. To improve its production safety, d-alanine-deficient heterologous gene expression systems were constructed without antibiotics. The integrated expression and co-expression plasmids were used in different systems, also exploiting the need for d-alanine during cellular metabolism. The integration of the β-galactosidase gene in recombinant is uniquely innovative and promising, applying common knockout techniques to the expression of target genes and the production of high-value products.d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

Highlights

D -tagatose is a popular monosaccharide with sweetness similar to sucrose but has30% fewer calories, possesses probiotic properties that improve intestinal flora, and has desirable coloring properties
The thermosensitive pTSC plasmid was introduced into the two D-alanine racemase knockout hosts to eliminate the introduced bleomycin resistance gene, yielding B. subtilis 168 D1P2 and B. subtilis 168 D2P2
Strains that did not grow on plates containing D-alanine and bleomycin and kanamycin but did grow on D-alanine plates containing no antibiotic were identified as recombinant B. subtilis lacking D-alanine and constituted the first step in validating the recombinant construct (Figure 1)

Summary

Introduction

D -tagatose is a popular monosaccharide with sweetness similar to sucrose but has30% fewer calories, possesses probiotic properties that improve intestinal flora, and has desirable coloring properties. D-tagatose is an optimal alternative sweetener and is widely used in healthy foods [1,2]. D-tagatose has been recognized as safe (GRAS) by the US Food and Drug Administration 2001) [3] and was approved as a safe food in 2004 by the Joint Expert Committee on. The global artificial sweetener market, estimated to be $3.2 billion in 2016 [4], is expected to expand, and so safe and high-yield production of D-tagatose has become a pressing problem. After sequencing the whole genome of Bacillus subtilis, the integration vector technique expanded, with B. subtilis becoming the most widely used microorganism in industry. Integrated plasmids used to insert exogenous genes can carry resistance genes, restricting their use in the food industry. An antibiotic-free integrated expression system was designed using a CRE/loxP-specific recombination system and D-alanine racemase activity

Methods

Results

Discussion

Conclusion