Isolation and Screening of High-Yielding α-Amylase Mutants of Bacillus subtilis by Heavy Ion Mutagenesis.

To improve fermentative production of α-amylase, heavy-ion mutagenesis technology was used to irradiate Bacillus subtilis (B. subtilis) to obtain the high yielding mutants in this study. After continuous cultivation for 12 generations, eight mutants exhibited positive mutation rate with greater H/C. The α-amylase production was stable and obviously exceeded that by the parent strain, which shows that the mutants have a good genetic stability. Among the mutants, the α-amylase activity of B. subtilis KC-180-2 was 72.26 U·mL-1, which was 82.34% higher than that of the original strain. After optimization of fermentation conditions and media, the α-amylase activity of B. subtilis KC-180-2 reached a maximum of 156.83 U·mL-1 at 36 h in a bioreactor. In addition, the optimized fermentation temperature of B. subtilis KC-180-2 was increased to 49℃, indicating B. subtilis KC-180-2 possesses high-temperature resistance, which has great application prospects for industrial fermentation for α-amylase production.

Overexpression of enzymes in glycolysis and energy metabolic pathways to enhance coenzyme Q10 production in Rhodobacter sphaeroides VK-2-3.

We subjected the components of the glycolysis and energy metabolism pathways of Rhodobacter sphaeroides (R. sphaeroides) to metabolic engineering to improve the titer and yield of coenzyme Q10 (CoQ10). Phosphofructokinase (PFK), cyclic adenylate-dependent protein kinase (PKAC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and adenosine triphosphate hydrolase (KdpC) were overexpressed in R. sphaeroides VK-2-3 (VK-2-3). The strains were labeled R. sphaeroides PFK (RS.PFK), RS.PKAC, RS.PFK-PKAC, RS.KdpC, RS.GAPDH, and RS.KdpC-GAPDH. Results showed that the CoQ10 titers of RS.PFK, RS.PKAC, and RS.PFK-PKAC were 300.96 ± 0.87, 405.94 ± 4.77, and 379.94 ± 0.42 mg/l, respectively. The CoQ10 titers of RS.PFK and VK-2-3 were not significantly different; however, those for RS.PKAC and RS.PFK-PKAC were 13 and 6% higher than that of VK-2-3, respectively. Further, the titers of RS.KdpC, RS.GAPDH, and RS.KdpC-GAPDH were 360.17 ± 0.39, 409.79 ± 0.76, and 359.87 ± 1.14 mg/l, respectively. The titers of RS.KdpC and RS.KdpC-GAPDH were not significantly different from that for VK-2-3, whereas that for RS.GAPDH was 14% higher than that of VK-2-3. Finally, when the cultures of RS.GAPDH and VK-2-3 were scaled up in 5-L fermenters, the CoQ10 titers and RS.GAPDH yields increased by 44.3 and 37.8%, respectively, compared with VK-2-3.To the best of our knowledge, the glycolysis pathway of R. sphaeroides was studied for the first time in this study. We genetically modified the components of the energy metabolism pathway to obtain the strain with high yield of CoQ10 mutant RS.GAPDH. The findings of this study can serve as a basis for future studies involving metabolic engineering of CoQ10-producing strains.

An isolated cellulolytic Escherichia coli from bovine rumen produces ethanol and hydrogen from corn straw.

BACKGROUND: Lignocellulosic biomass is the most abundant resource on earth. Lignocellulose is mainly composed of cellulose, hemicelluloses, and lignin. The special construction of three kinds of constituents led to the prevention of effective degradation. The goal of this work was to investigate the great potentials of bovine rumen for novel cellulolytic bacterial isolation, which may be used for chemicals and biofuel production from lignocellulose. RESULTS: A cellulolytic strain, ZH-4, was isolated from Inner Mongolia bovine rumen. This strain was identified as Escherichia coli by morphological, physiological, and biochemical characteristics and 16S rDNA gene sequencing. The extracellular enzyme activity analysis showed that this strain produces extracellular cellulases with an exoglucanase activity of 9.13 IU, an endoglucanase activity of 5.31 IU, and a ß-glucosidase activity of 7.27 IU at the pH 6.8. This strain was found to produce 0.36 g/L ethanol and 4.71 mL/g hydrogen from corn straw with cellulose degradation ratio of 14.30% and hemicellulose degradation ratio of 11.39%. CONCLUSIONS: It is the first time that a cellulolytic E. coli was isolated and characterized form the bovine rumen. This provided a great opportunity for researchers to investigate the evolution mechanisms of the microorganisms in the rumen and provided great chance to produce biofuels and chemicals directly from engineered E. coli using consolidated bioprocess.

Production of novel microbial flocculants by Klebsiella sp. TG-1 using waste residue from the food industry and its use in defecating the trona suspension.

A microbial-flocculants-producing (MBF-producing) bacterium, named TG-1, was isolated from waste water of a starch factory, and identified as Klebsiella sp. TG-1. The microbial flocculants (MBF) produced by TG-1, named as MBF-TG-1, was applied to defecating the strong basic trona suspension in the trona industry. After optimizing medium and culturing conditions with single-factor and orthogonal designs, the highest flocculation rate of 86.9% was achieved. Chemical analysis showed that the purified microbial flocculants (MBF-TG-1) was mainly composed of polysaccharides (84.6%), with a small amount of protein or amino acid (11.1%). Bridging mechanism was supposed as the main flocculation mechanism by analyzing the flocculation process and the biochemistry properties of MBF-TG-1. The high flocculation rate (84%) was also achieved with a low-cost medium (the solid residue of tofu production from food industry).