Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

Abstract

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 (SPLc), 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 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 3 L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6 mmol L−1 ZnCl2 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 SPLc 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

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

“…LMG 10502 E. coli Rosetta 2 1.5 U/mL cell culture (OD 1) (~ 6%) Aerts et al [ 37 ] B. a. DSM 20083 E. coli BL21(DE3) 38 U/mg 33% This study LmSP L. m. DSM 20193 L. m. DSM 20193 7 U/mg 3.7% Koga et al [ 38 ] E. coli DH10B 48 U/mg 25% Goedl et al [ 28 ] E. coli BL21(DE3) 55 U/mg 30% This study L. m. ATCC 12291 E. coli 1101 (slp- spl -1) 55.7 U/mg 30% Kitoa et al [ 39 ] L. m. No. 165 E. coli JM109 4.6 U/mg 50% Kawasaki et al [ 40 ] L.m E. coli BL21(DE3) ~ 22 U/mg CDM (~ 44 U/mg CFE) 25% Su et al [ 41 ] L. m. NRRL B-1149 E . coli BL21(DE3)pLysS 1.49 U/mg Specific activities of enzymes Lee et al [ 43 ] L. m. NRRL B-742 E. coli BL21(DE3)pLysS 1.8 U/mg Lee et al [ 42 ] …”