Xiaoming Bao

Status and prospect of lignocellulosic bioethanol production in China

As a developing country with the largest population, China faces a serious challenge in satisfying its continuously increasing energy demand, especially for liquid fuel. Bioethanol production from lignocellulosic material is a potential and feasible method to solve the many problems in China, and it was supported by the Chinese government. Many research projects in China on lignocellulosics ethanol production have been carried out. After more than 30years of research, several pilot scale facilities have been constructed. This review focuses on the recent research activities and developments in lignocellulosic ethanol production during the past decade in China. As case study, a corncob biorefinery process is introduced.

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

Aims:  To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae.

Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae

Improving the cellulase secretion is beneficial for Saccharomyces cerevisiae used in consolidated bioprocessing (CBP) of cellulosic ethanol. In this study, protein secretory pathway, including protein folding, disulfide bond formation, and protein trafficking and sorting, was modified in S. cerevisiae. The effects of these modifications on the secretion of cellobiohydrolase I (Tr-Cel7A) with its native signal peptide from Trichoderma reesei were investigated. The results showed that overexpression of the protein disulfide isomerase Sc-PDI1 and the plasma membrane targeting soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sc-SSO1, and disruption of the sorting receptor Sc-VPS10 and a Ca(2+)/Mn(2+) ATPase Sc-PMR1, improved respectively the extracellular Tr-Cel7A activities. Among them, disruption of Sc-PMR1 showed better improvement of 162% in the extracellular activity and decreased the glycosylation of Tr-Cel7A. Multiple modifications generally resulted in higher activities. The extracellular activities of the quadruple-modified strain (vps10Δ/pmr1Δ/SSO1/PDI1/cel7AF) using p-nitrophenyl-β-d-cellobioside (pNPC) and phosphoric acid swollen cellulose (PASC) as the substrates, respectively, were 3.9-fold and 1.3-fold higher than that of the reference strain cel7AF. The results indicated that engineering of the protein secretory pathway is an effective approach to improve the Tr-Cel7A secretion in S. cerevisiae.

Ethanolic cofermentation with glucose and xylose by the recombinant industrial strain Saccharomyces cerevisiae NAN-127 and the effect of furfural on xylitol production.

Saccharomyces cerevisiae strain NAN-127 (2n, prototroph), which contains the xylose reductase-xylitol dehydrogenase (XR-XDH) xylose metabolic pathway was used for the cofermentation of glucose and xylose. Oxygen supply was the most important factor for xylose fermentation and pH 4.5 and a ventilation rate of 0.04 vvm were optimal. The xylose utilization ratio reached 0.655 at an initial xylose concentration of 50 gL(-1) and was 0.9 at an initial concentration of 20 gL(-1). Addition of furfural at late logarithmic phase as electron acceptor to a final concentration of 3.0 gL(-1) decreased the xylitol yield by 17% under micro-aeration conditions without inhibiting cell growth, but also without an increase in ethanol yield. The results are important to the application of strain NAN-127 in the lignocellulosic ethanol process.

Genetic and comparative transcriptome analysis of bromodomain factor 1 in the salt stress response of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae BDF1 gene, which encodes a bromodomain-containing transcription factor, was previously isolated by transposon mutugenesis in a screen for salt-sensitive mutants. However, the salt stress response mechanism regulated by bromodomain transcription factor 1 protein (Bdf1p) remains poorly understood. In this report, genetic analysis indicated that the salt sensitivity of the BDF1 deletion mutant was suppressed by increased gene dosage of its homologous gene BDF2. Furthermore, comparative transcriptome analysis revealed that the differences in transcriptional response between the wild type and the bdf1Δ mutant in the presence of salt stress (0.6 mol/L NaCl, 45 min) were mainly related to cell-wall biosynthesis, the mitochondria, and several unknown genes. Our results provided further information about the regulatory mechanism involved in the salt stress response and adds new insight for understanding the biological functional of bromdomain-containing proteins in cellular processes.

Improvement of L-Arabinose Fermentation by Modifying the Metabolic Pathway and Transport in Saccharomyces cerevisiae

The L-arabinose utilization pathway was established in Saccharomyces cerevisiae, by expressing the codon-optimized araA, araB, and araD genes of Lactobacillus plantarum. After overexpressing the TAL1, TKL1, RPE1, RKI1, and GAL2 genes and adaptive evolution, the L-arabinose utilization of the recombinant strain became efficient. The resulting strain displayed a maximum specific growth rate of 0.075 h−1, a maximum specific L-arabinose consumption rate of 0.61 g h−1 g−1 dry cell weight, and a promising ethanol yield of 0.43 g g−1 from L-arabinose fermentation.

Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second‐generation bioethanol production

To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild‐type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h−1 in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g−1 consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production.

Cloning a Cuticle-Degrading Serine Protease Gene with Biologic Control Function from Beauveria brongniartii and Its Expression in Escherichia coli

Beauveria brongniartii extracellular subtilisin-like serine protease (Pr1) is one of the most virulent factors by virtue of its activity against insect cuticles. The Pr1 cDNA was cloned using the switching mechanism at the 5′ end of the RNA transcript and rapid amplification of cDNA ends. The 1732-bp fragment of genomic DNA containing the predicted open-reading frame of the Pr1 gene was cloned by polymerase chain reaction and sequenced. The Pr1 cDNA is 1550 bp and contains an 1140-bp ORF. The deduced amino-acid sequence of the protein shows identity to that of proteinase K from Tritirachium album (62%), Pr1 from Metarhizium nisopliae (67%), and Pr1 from B. bassiana (76%). The Pr1 protein with an N-terminal fusion to the six-histidine tag was expressed in Escherichia coli as inclusion bodies with the expression vector pBV220. Sodium dodecylsulsulfate–polyacrylamide gel electrophoresis clearly revealed expressed product. The Pr1 protein was purified and refolded and had proteolytic activity of 0.288 U mg−1.