Development of a thiostrepton-free system for stable production of PLD in Streptomyces lividans SBT5.

BACKGROUND: Phospholipase D (PLD) is highly valuable in the food and medicine industries, where it is used to convert low-cost phosphatidylcholine into high-value phospholipids (PLs). Despite being overexpressed in Streptomyces, PLD production requires expensive thiostrepton feeding during fermentation, limiting its industrialization. To address this issue, we propose a new thiostrepton-free system. RESULTS: We developed a system using a combinatorial strategy containing the constitutive promoter kasOp* and PLD G215S mutation fused to a signal peptide sigcin of Streptoverticillium cinnamoneum pld. To find a candidate vector, we first expressed PLD using the integrative vector pSET152 and then built three autonomously replicating vectors by substituting Streptomyces replicons to increase PLD expression. According to our findings, replicon 3 with stability gene (sta) inserted had an ideal result. The retention rate of the plasmid pOJ260-rep3-pld* was 99% after five passages under non-resistance conditions. In addition, the strain SK-3 harboring plasmid pOJ260-rep3-pld* produced 62 U/mL (3.48 mg/g) of PLD, which further improved to 86.8 U/mL (7.51 mg/g) at 32 °C in the optimized medium, which is the highest activity achieved in the PLD secretory expression to date. CONCLUSIONS: This is the first time that a thiostrepton-free PLD production system has been reported in Streptomyces. The new system produced stable PLD secretion and lays the groundwork for the production of PLs from fermentation stock. Meanwhile, in the Streptomyces expression system, we present a highly promising solution for producing other complex proteins.

Alkalophilic adaptation of XynB endoxylanase from Aspergillus niger via rational design of pKa of catalytic residues.

Based on the strategy of changing pH-stability profiles by altering pKa values of catalytic residues, rational protein engineering was applied to improve alkalophilic adaptation of Aspergillus niger endoxylanase XynB. Computational predictions and molecular modeling were carried out using PROPKA server and SWISS-MODEL server, respectively. Three endoxylanase mutant of S108V, N151E, and Q178R, in which the pKa values of either catalytic glutamate residues shifted, were generated. In agreement with expectation, the variant of Q178R improved alkalophilic performances. The mutant Q178R raised the optimum pH of XynB from 5.5 to 6.0 and retained 37% of the maximum activity at pH 8.0. Interestingly, the pKa values of Glu84 and Glu175 in Q178R are 7.91 and 6.32, respectively. The pKa of Glu175 is lower than that of Glu84, as opposed to the fact that the pKa of Glu84 is lower than that of Glu175 in other GH11 xylanases. It indicated that Glu175 may convert into a nucleophile residue and Glu84 into an acid/base residue.