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

Abstract

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.

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast

“…Many d -tagatose production routes involve epimerization, including the formation of cheap dairy by-products (cheese whey powder, etc.) or the upstream intermediate disaccharide lactose [ 98 ]. l -arabinose isomerases ( l -AIs) can convert d -galactose to d -tagatose.…”

Advances and Prospects of d-Tagatose Production Based on a Biocatalytic Isomerization Pathway

“…In addition, antibiotics are not allowed in the food industry. Zhang et al [100] successfully produced D-tagatose in recombinant Bacillus subtilis whole cells by constructing a heterologous gene expression system that did not require additional antibiotics. These methods are expected to be more widely used in the food industry in the future to meet safety and quality requirements.…”

Novel In Vitro Multienzyme Cascade for Efficient Synthesis of d-Tagatose from Sucrose

“…The enzymatic biosynthesis of D-tagatose has become a major interest in research recently due to its low cost, high purity, and environmental friendliness. For instance, a two-enzyme (β-galactosidase and L-arabinose isomerase) catalysis has poor enzyme specificity and a low conversion (less than 45%) for D-tagatose synthesis using lactose as a substrate [11,12]. Approximately 72% conversion was obtained after galactosol-2-dehydrogenase derived from Rhizobium leguminosarum bv.…”