Xiao-Zhou Zhang

Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

ABSTRACT We developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, and BL21(DE3)] and Bacillus subtilis for obtaining chimeric plasmids.

One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis

Although intensive efforts have been made to create recombinant cellulolytic microorganisms, real recombinant cellulose-utilizing microorganisms that can produce sufficient secretory active cellulase, hydrolyze cellulose, and utilize released soluble sugars for supporting both cell growth and cellulase synthesis without any other organic nutrient (e.g., yeast extract, peptone, amino acids), are not available. Here we demonstrated that over-expression of Bacillus subtilis endoglucanase BsCel5 enabled B. subtilis to grow on solid cellulosic materials as the sole carbon source for the first time. Furthermore, two-round directed evolution was conducted to increase specific activity of BsCel5 on regenerated amorphous cellulose (RAC) and enhance its expression/secretion level in B. subtilis. To increase lactate yield, the alpha-acetolactate synthase gene (alsS) in the 2,3-butanediol pathway was knocked out. In the chemically defined minimal M9/RAC medium, B. subtilis XZ7(pBscel5-MT2C) strain (ΔalsS), which expressed a BsCel5 mutant MT2C, was able to hydrolyze RAC with cellulose digestibility of 74% and produced about 3.1g/L lactate with a yield of 60% of the theoretical maximum. When 0.1% (w/v) yeast extract was added in the M9/RAC medium, cellulose digestibility and lactate yield were enhanced to 92% and 63% of the theoretical maximum, respectively. The recombinant industrially safe cellulolytic B. subtilis would be a promising consolidated bioprocessing platform for low-cost production of biocommodities from cellulosic materials.

Enhanced Microbial Utilization of Recalcitrant Cellulose by an Ex Vivo Cellulosome-Microbe Complex

ABSTRACT A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

ABSTRACT A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases.

A synthetic enzymatic pathway was designed for the deep oxidation of glucose in enzymatic fuel cells (EFCs). Polyphosphate glucokinase converts glucose to glucose-6-phosphate using low-cost, stable polyphosphate rather than costly ATP. Two NAD-dependent dehydrogenases (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) that were immobilized on the bioanode were responsible for generating two NADH per glucose-6-phosphate (i.e., four electrons were generated per glucose via a diaphorase-vitamin K(3) electron shuttle system at the anode). Additionally, to prolong the enzyme lifetime and increase the power output, all of the recombinant enzymes that originated from thermophiles were expressed in Escherichia coli and purified to homogeneity. The maximum power density of the EFC with two dehydrogenases was 0.0203 mW cm(-2) in 10 mM glucose at room temperature, which was 32% higher than that of an EFC with one dehydrogenase, suggesting that the deep oxidation of glucose had occurred. When the temperature was increased to 50°C, the maximum power density increased to 0.322 mW cm(-2), which was approximately eight times higher than that based on mesophilic enzymes at the same temperature. Our results suggest that the deep oxidation of glucose could be achieved by using multiple dehydrogenases in synthetic cascade pathways and that high power output could be achieved by using thermostable enzymes at elevated temperatures.

Glycoside hydrolase family 9 processive endoglucanase from Clostridium phytofermentans: heterologous expression, characterization, and synergy with family 48 cellobiohydrolase.

The glycoside hydrolase family 9 cellulase (Cel9) from Clostridium phytofermentans has a multi-modular structure and is essential for cellulose hydrolysis. In order to facilitate production and purification of Cel9, recombinant Cel9 was functionally expressed in Escherichia coli. Cel9 exhibited maximum activity at pH 6.5 and 65 degrees C on carboxymethyl cellulose in a 10-min reaction period. The hydrolysis products on regenerated amorphous cellulose (RAC) were cellotetraose (a major product), cellotriose, cellobiose and glucose, and 71-80% of the reducing sugars produced by Cel9 were in soluble form, suggesting that Cel9 was a processive endoglucanase. The highest synergy between C. phytofermentans Cel9 and C. phytofermentans cellobiohydrolase Cel48 on Avicel was about 1.8 at a ratio of about 1:5. Cel9 alone was sufficient to solublize filter paper while Cel48 was not; however, it enhanced the solublization process along with Cel9 synergistically. This study provided useful information for understanding of the cellulose hydrolysis mechanism of this cellulolytic bacterium with potential industrial importance.

Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.

The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.

Transformation of Bacillus subtilis

Bacillus subtilis has tremendous applications in both academic research and industrial production. However, molecular cloning and transformation of B. subtilis are not as easy as those of Escherichia coli. Here we developed a simple protocol based on super-competent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids generated by prolonged overlap extension-PCR. Super-competent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. This new protocol is simple (e.g., restriction enzyme, phosphatase, and ligase free), fast, and highly efficient (i.e., ~10(7) or ~10(4) transformants per μg of multimeric plasmid or ligated plasmid DNA, respectively). Shuttle vectors for E. coli-B. subtilis are not required.