Combining Cell Surface Display and DNA-Shuffling Technology for Directed Evolution of Streptomyces Phospholipase D and Synthesis of Phosphatidylserine

Abstract

Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.

Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis

“…subtilis WB800/pMA5-sfGFP-PLD R34 and WB800/pP43NMK-NprB-PLD R34 were more suitable for the expression of Recom-34 than E. coli …”

“…These results exceeded the 80.3% conversion rate of E. coli expressed Recom-34 in the diethyl ether reaction system . In the case of ethyl acetate, the presence of substrates and products could not be detected, indicating a low solubility of the substrate in this solvent.…”

“…26 This finding can also be correlated to the use of a new protein secretion system mediated by a super-folder fluorescent protein (EGPSS) developed in E. coli, which uses the GFP as the N-terminal leading protein to guide heterologous proteins out of the cells from the cytoplasm. 27 In our previous studies, an engineered Streptomyces PLD (Recom-34) after DNA shuffling was obtained, which also showed an excellent performance in the E. coli expression system. 28 In this study, we constructed an efficient and visualized secretory expression system for Recom-34 expression in B. subtilis.…”

“…In our previous studies, an engineered Streptomyces PLD (Recom-34) after DNA shuffling was obtained, which also showed an excellent performance in the E. coli expression system . In this study, we constructed an efficient and visualized secretory expression system for Recom-34 expression in B.…”

Bacillus velezensis EEAM 10B Strengthens Nutrient Metabolic Process in Black Soldier Fly Larvae (Hermetia illucens) via Changing Gut Microbiome and Metabolic Pathways

“…The QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) was used to extract the gDNA in both sets. The 16S rRNA gene was amplified using universal 1492R/27F primers ( 33 ).…”