BACKGROUNDWe 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 (SPLc), a naturally cytoplasmic enzyme, exhibits excellent properties for synthesis of -arbutin, but no systemic investigation of its preparation has thus far been reported. RESULTSThree 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 SPLc was co-expressed with these PLCs, and the location of the PLC and SPLc genes in the co-expression vector was optimized, the highest production was obtained when SPLc gene was inserted upstream of the B.cereus PLC gene, as in BL21(DE3)/pETDuet-sp-bcplc. SPLc production was then scaled up to a 3L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6mmolL(-1) ZnCl2 and protein expression was induced at a dry cell weight of 15gL(-1) by the addition of lactose at 0.2gL(-1)h(-1) at 30 degrees C, the extracellular SPLc activity reached its highest level of 1445.1UmL(-1) This constituted 81.6% of total activity; the remainder of the enzyme (18.4%) remained inside the cells. CONCLUSIONThis 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. (c) 2018 Society of Chemical Industry