Discovery and functional verification of endogenous glucanases for scleroglucan hydrolysis in Sclerotium rolfsii / 生物工程学报

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.

Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli / 生物工程学报

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.

High-throughput screening of Methylobacterium extorquens for high production of pyrroloquinoline quinone / 生物工程学报

Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could be applied in food, medicine and other fields. Due to the extremely high cost of chemical synthesis, the production of PQQ by microbial fermentation attracted more and more attention. At present, the production titer of PQQ by fermentation method is too low to achieve industrial application. Due to the lack of a thorough understanding of the PQQ biosynthesis and its regulation mechanisms, and the lack of necessary genetic engineering modification methods for wild type strains, metabolic engineering of microorganisms to enhance PQQ production still lacks essential requirements. In this study, a PQQ-producing bacterium, Methylobacterium extorquens I-F2, was employed as a model strain. By integration of Atmospheric and room temperature plasma (ARTP) mutagenesis, flow cytometry sorting and high-throughput screening strategies, optimization of sample preparation and flow sorting process, a high-titer PQQ mutant strain was obtained. The titer of PQQ was increased by 98.02% compared with that of M. extorqunens I-F2. The process described here showed that the combination of the flow cytometry with high-throughput screening method can be used to obtain high-titer mutants more simply and rapidly, compared with genetic engineering and traditional screening methods.