Zhenghua Gu

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ(max)) could reach 0.03 and 0.05 h(-1) under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobioseL(-1) and produced 2.3 g ethanol L(-1) in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L(-1) and produced 1.5 g ethanolL(-1) over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11h(-1)) on cellobiose.

Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran

Three strains, Bacillus subtilis B1, B. subtilis B18 and Bacillus thuringiensis B12, were screened from wheat bran to produce milk-clotting enzyme. Among them, B. subtilis B1 exhibited considerable milkclotting activity with low proteolytic activity. After response surface methodology optimization, milkclotting activity was improved from 782 SU ml -1 to 1129.05 ± 74.55 SU mL -1 with a small inoculum size (0.130%, v/v). The optimized medium contained glucose (16.2 gL -1 ), wheat bran (30 gL -1 ), NaCl (5 gL -1 ), MgSO 4 .7H 2 O (5 gL -1 ), KH 2 PO 4 (2 gL -1 ) and CaCO 3 (3 gL -1 ). The milk-clotting enzyme from B. subtilis B1 has the optimum pH 5.5 and CaCl 2 concentration 50 mmol L -1 . It is completely inactivated after 5 min at 70°C. The result showed that B. subtilis B1 was a promising strain for industrial milk-clotting enzyme production. Key words : Milk-clotting enzyme, Bacillus subtilis, submerged fermentation, wheat bran.

Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes.

We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS.

Screening, characterization and utilization of D-amino acid aminotransferase to obtain D-phenylalanine

D-phenylalanine (D-Phe) is an important side-chain building block for semi-synthetic penicillins and cephalosporins. To synthesize D-Phe, D-amino acid aminotransferases (Dat) from Bacillus subtilis WB600, Bacillus licheniformis ATCC 14580, and Bacillus amyloliquefaciens were firstly compared. The theoretical 3 dimensional models of Dat were constructed, which showed appropriate configuration for synthesis of D-Phe in virtual screening. The comparison 3 different Dat on D-Phe formation was tested. All the heterogenous Dat showed high thermostability and pH stability, while Dat from B. subtilis demonstrated a better potential for Phe production than those from B. licheniformis and B. amyloliquefaciens. Then the dat from B. subtilis was expressed in an L-Phe producing chassis of Escherichia coli W14 (pR15ABK) to obtain D-Phe and different factors affecting D-Phe accumulation were tested. The engineered strain E. coli BCEA (pR15ABKApRdatBS) accumulated 1.72 g/L D-Phe in a 15-L jar fermenter which is the highest fermentation concentration that had been reported.

Effects of mixed carbon sources on galactose and mannose content of exopolysaccharides and related enzyme activities in Ganoderma lucidum

This work investigated the effects of mixed carbon sources on the monosaccharide composition of exopolysaccharides (EPS) and activity of related enzymes, phosphoglucose isomerase (PGI), α-phosphoglucomutase (PGM), phosphomannose isomerase (PMI) and GDP-D-Man pyrophosphorylase (GMP), in the EPS biosynthesis by Ganoderma lucidum. Combinations of two carbon sources, glucose (Glc) + mannose (Man) and Glc + galactose (Gal), were applied at various mass ratios in the liquid culture of G. lucidum. The results showed that Glc, Gal, and Man were the major monosaccharides in G. lucidum EPS, and the combined mole percentages of monosaccharides were >80%, the same as those in the carbon source mixture. Activities of PGI, PMI, and GMP were correlated with Man mole percentage in EPSs and were enhanced by increasing the proportion of Gal for the carbon source Glc + Gal. PGM activity was correlated with Gal mole percentage and was reduced by increasing the Man proportion for Glc + Man. The expression of four genes encoding PGI, PMI, GMP and PGM reached peak levels on day 6 of culture, whereas the enzyme activities increased steadily from day 3 to 8. The monosaccharide composition of G. lucidum EPS for various mixed carbon source conditions thus appeared to be controlled by the translational level of genes encoding PGM, PGI, PMI, and GMP. These findings will be helpful to control the monosaccharide composition for desired biological activity of EPS produced by G. lucidum fermentation.

Development of an inducible secretory expression system in Bacillus licheniformis based on an engineered xylose operon

The xylose operon can be an efficient biological component for regulatory expression uses in Bacillus licheniformis. However, its characteristic susceptibility to carbon catabolite repression (CCR) makes its application inconvenient. In this study, plasmids harboring the wild-type operons from three Bacillus species were constructed and introduced into B. licheniformis. These plasmids ensured secretory expression of maltogenic α-amylase (BLMA) in B. licheniformis under strict regulation. The glucose-mediated CCR was then alleviated by engineering the xylose operon of the expression system. Evidence showed that mutations in the highly conserved nucleotides of the identified catabolite responsive element (cre) consensus sequence prevented association of the regulator CcpA with DNA, thus resulting in an increase in BLMA activity of up to 12-fold. Furthermore, features of this engineered system for inducible expression were investigated. Induction in mid-log phase using 10 g/L xylose at 37 °C was found to be beneficial for promoting the accumulation of recombinant product, and the maximum yield of BlmMA reached 715.4 U/mL. This study contributes to the industrial application of the generally recognized as safe (GRAS) workhorse B. licheniformis.