Qinhong Wang

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2.

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl(2), a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g(-1), which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.

Chromosome condensation in the absence of the non-SMC subunits of MukBEF.

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation.

Ionic liquids-based hydrolysis of Chlorella biomass for fermentable sugars.

An ionic liquids-based chemical hydrolysis strategy was developed to obtain high-yielding soluble sugars from Chlorella biomass. Initial ionic liquids dissolution and subsequently HCl catalyzed hydrolysis could dissolve 75.34% of Chlorella biomass and release 88.02% of total sugars from Chlorella biomass. The amount of HCl loading was 7 wt.% relative to Chlorella biomass weight, which was much lower (only 14.6%) than that in HCl/MgCl(2)-catalyzed system with similar sugars release (Zhou et al., 2011). Ionic liquids in the hydrolysates were recycled and fermentable sugars were evaluated by converting to bioethanol after separated by ion-exclusion chromatography. This ionic liquids-based hydrolysis strategy showed the great potential to produce fermentable sugars from algal biomass.

MukEF Is Required for Stable Association of MukB with the Chromosome

MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.

Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering.

The Alcohol Dehydrogenase System in the Xylose-Fermenting Yeast Candida maltosa

Background The alcohol dehydrogenase (ADH) system plays a critical role in sugar metabolism involving in not only ethanol formation and consumption but also the general “cofactor balance” mechanism. Candida maltosa is able to ferment glucose as well as xylose to produce a significant amount of ethanol. Here we report the ADH system in C. maltosa composed of three microbial group I ADH genes (CmADH1, CmADH2A and CmADH2B), mainly focusing on its metabolic regulation and physiological function. Methodology/Principal Findings Genetic analysis indicated that CmADH2A and CmADH2B tandemly located on the chromosome could be derived from tandem gene duplication. In vitro characterization of enzymatic properties revealed that all the three CmADHs had broad substrate specificities. Homo- and heterotetramers of CmADH1 and CmADH2A were demonstrated by zymogram analysis, and their expression profiles and physiological functions were different with respect to carbon sources and growth phases. Fermentation studies of ADH2A-deficient mutant showed that CmADH2A was directly related to NAD regeneration during xylose metabolism since CmADH2A deficiency resulted in a significant accumulation of glycerol. Conclusions/Significance Our results revealed that CmADH1 was responsible for ethanol formation during glucose metabolism, whereas CmADH2A was glucose-repressed and functioned to convert the accumulated ethanol to acetaldehyde. To our knowledge, this is the first demonstration of function separation and glucose repression of ADH genes in xylose-fermenting yeasts. On the other hand, CmADH1 and CmADH2A were both involved in ethanol formation with NAD regeneration to maintain NADH/NAD ratio in favor of producing xylitol from xylose. In contrast, CmADH2B was expressed at a much lower level than the other two CmADH genes, and its function is to be further confirmed.

Screening of pyruvate-producing yeast and effect of nutritional conditions on pyruvate production.

Aims: To find a yeast strain that can overproduce pyruvate and to investigate the effect of nutrients on pyruvate production.

High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

Aims:  To develop a high‐throughput assay for screening xylose‐utilizing and ethanol‐tolerant thermophilic bacteria owing to their abilities to be the promising ethanologens.

Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

Branched-chain higher alcohols.

Chinas energy requirements and environmental concerns have stimulated efforts toward developing alternative liquid fuels. Compared with fuel ethanol, branched-chain higher alcohols (BCHAs), including isopropanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, exhibit significant advantages, such as higher energy density, lower hygroscopicity, lower vapor pressure, and compatibility with existing transportation infrastructures. However, BCHAs have not been synthesized economically using native organisms, and thus their microbial production based on metabolic engineering and synthetic biology offers an alternative approach, which presents great potential for improving production efficiency. We review the current status of production and consumption of BCHAs and research progress regarding their microbial production in China, especially with the combination of metabolic engineering and synthetic biology.