Construction and co-expression of polycistronic plasmids encoding thermophilic l-arabinose isomerase and hyperthermophilic β-galactosidase for single-step production of d-tagatose

• Published in: Biochemical engineering journal 2016-05, Vol.109, p.28-34
• Main Authors: Xu, Zheng, Xu, Zhaoxian, Tang, Bao, Li, Sha, Gao, Jian, Chi, Bo, Xu, Hong
• Format: Article
• Language: English
• Subjects: Biocatalysis; Bioprocess design; d-Tagatose; Enzymes; Escherichia coli; l-Arabinose isomerase; Lactobacillus fermentum; Lactose; Thermus thermophilus
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2015.12.015

[Display omitted] •AI from L. fermentum and β-galactosidase from T. thermophilus were co-expressed.•101g/L l-ribose with yield of 20.2% and productivity of 6.3g/Lh was obtained.•The orders of the encoding genes, SD, and AS length were optimized.•Cells showed balanced activity for two enzymes using optimized SD and AS sequence. d-Tagatose is a naturally occurred hexoketose. It is rarely found in nature and shows advantages as a low calorie sweetener and potentials as the substitute of sucrose. The biological production of d-tagatose involves hydrolysis of lactose by β-galactosidase and isomerization of released d-galactose into d-tagatose by l-arabinose isomerase (AI). In this study, two thermostable enzymes including an AI from Lactobacillus fermentum CGMCC2921 and a β-galactosidase from Thermus thermophilus HB27 were co-expressed in Escherichia coli. The order of the two genes, the downstream Shine–Dalgarno (SD) sequence and the aligned spacing (AS) length were optimized to balance their expression levels. One recombinant E. coli strain showed balanced activities for the two enzymes using GAAGGAGA as the downstream SD sequence and 10 nucleotides (nt) for AS. During whole cell bio-catalysis, the recombinant E. coli cells showed optimal catalytic temperature and pH at 70°C and 7.0, respectively. With the addition of borate, d-tagatose was produced directly from cheap raw material lactose in 16h in a concentration of 101g/L, a yield of 20.2%, and a productivity of 6.3g/Lh. This study shed light on the efficient and economical single-step production of d-tagatose in a thermophilic bio-conversion system.