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.

Cloning, purification and biochemical characterisation of a GH35 beta-1,3/beta-1,6-galactosidase from the mucin-degrading gut bacterium Akkermansia muciniphila.

A putative GH35 ß-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74 kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42 °C, and the optimum pH was determined to be pH 3.5. The addition of sodium dodecyl sulphate (SDS) reduced the enzyme's activity significantly. The addition of Mg2+-ions decreased the activity of the ß-galactosidase, whereas other metal ions or EDTA showed no inhibitory effect. The enzyme catalysed the hydrolysis of ß1,3- and ß1,6- linked galactose residues from various substrates, whereas only negligible amounts of ß1,4-galactose were hydrolysed. The present study describes the first functional characterisation of a ß-galactosidase from this human gut symbiont.