Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

BACKGROUND We previously showed that extracellular production of naturally cytoplasmic proteins could be realized through co-expression with phospholipase C (PLC), which enhances cell membrane permeability through limited hydrolysis of membrane phospholipids. Leuconostoc mesenteroides sucrose phosphorylase (SP Lc ), a naturally cytoplasmic enzyme, exhibits excellent properties for synthesis of α-arbutin, but no systemic investigation of its preparation has thus far been reported. RESULTS Three PLCs were selected and expressed, without signal peptide, in Escherichia coli . More than 90% of the PLCs from Listeria monocytogenes and Bacillus cereus were present in the culture medium. When SP Lc was co-expressed with these PLCs, and the location of the PLC and SP Lc genes in the co-expression vector was optimized, the highest production was obtained when SP Lc gene was inserted upstream of the B. cereus PLC gene, as in BL21(DE3)/pETDuet -sp - bcplc . SP Lc production was then scaled up to a 3 L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6 mmol L −1 ZnCl 2 and protein expression was induced at a dry cell weight of 15 g L −1 by the addition of lactose at 0.2 g L −1 h −1 at 30 °C, the extracellular SP Lc activity reached its highest level of 1445.1 U mL −1 This constituted 81.6% of total activity; the remainder of the enzyme (18.4%) remained inside the cells. CONCLUSION This is the first report describing extracellular expression of sucrose phosphorylase, and the highest yield ever reported was obtained. This provides the basis for industrial-scale production and application of sucrose phosphorylase. It also provides a potential method for improving the production of other proteins and fine chemicals. © 2018 Society of Chemical Industry