Propionic acid, a major inhibitor to yeast cells, was accumulated during continuous ethanol fermentation from corn meal hydrolysate by the flocculating yeast under stillage backset conditions. Based on its inhibition mechanism in yeast cells, strategies were developed for alleviating this effect. Firstly, high temperature processes such as medium sterilization generated more propionic acid, which should be avoided. Propionic acid was reduced significantly during ethanol fermentation without medium sterilization, and concentrations of biomass and ethanol increased by 59.3% and 7.4%, respectively. Secondly, the running time of stillage backset should be controlled so that propionic acid accumulated would be lower than its half inhibition concentration IC50 (40 mmol/L). Finally, because low pH augmented propionic acid inhibition in yeast cells, a higher pH of 5.5 was validated to be suitable for ethanol fermentation under the stillage backset condition.
Biomass ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Propionates ; chemistry ; Yeasts ; metabolism

Industrial microorganisms are subject to various stress conditions, including products and substrates inhibitions. Therefore, improvement of stress tolerance is of great importance for industrial microbial production. Acetic acid is one of the major inhibitors in the cellulosic hydrolysates, which affects seriously on cell growth and metabolism of Saccharomyces cerevisiae. Studies on the molecular mechanisms underlying adaptive response and tolerance of acetic acid of S. cerevisiae benefit breeding of robust strains of industrial yeast for more efficient production. In recent years, more insights into the molecular mechanisms underlying acetic acid tolerance have been revealed through analysis of global gene expression and metabolomics analysis, as well as phenomics analysis by single gene deletion libraries. Novel genes related to response to acetic acid and improvement of acetic acid tolerance have been identified, and novel strains with improved acetic acid tolerance were constructed by modifying key genes. Metal ions including potassium and zinc play important roles in acetic acid tolerance in S. cerevisiae, and the effect of zinc was first discovered in our previous studies on flocculating yeast. Genes involved in cell wall remodeling, membrane transport, energy metabolism, amino acid biosynthesis and transport, as well as global transcription regulation were discussed. Exploration and modification of the molecular mechanisms of yeast acetic acid tolerance will be done further on levels such as post-translational modifications and synthetic biology and engineering; and the knowledge obtained will pave the way for breeding robust strains for more efficient bioconversion of cellulosic materials to produce biofuels and bio-based chemicals.
Acetic Acid ; pharmacology ; Genomics ; Industrial Microbiology ; Saccharomyces cerevisiae ; drug effects ; genetics

Zinc-finger proteins have been widely studied due to their highly conserved structures and DNA-binding specificity of zinc-finger domains. However, researches on the zinc-finger proteins from microorganisms, especially those from prokaryotes, are still very limited. This review focuses on the latest progress on microbial zinc-finger proteins, especially those from prokaryotes and the application of artificial zinc-finger proteins in the breeding of robust strains. Artificial zinc-finger proteins with transcriptional activation or repression domain can regulate the global gene transcription of microbial cells to acquire improved phenotypes, such as stress tolerance to heat, ethanol, butanol, and osmotic pressure. Using the zinc-finger domain as DNA scaffold in the construction of enzymatic system can enhance the catalytic efficiency and subsequently the production of specific metabolites. Currently, zinc-finger domains used in the construction of artificial transcription factor are usually isolated from mammalian cells. In the near future, novel transcription factors can be designed for strain development based on the natural zinc-finger domains from different microbes, which may be used to regulate the global gene expression of microbial cells more efficiently.
Bacteria ; metabolism ; DNA ; chemistry ; Protein Engineering ; Transcription Factors ; chemistry ; Transcriptional Activation ; Zinc Fingers

Microalgae have been identified as promising candidates for biorefinery of value-added molecules. The valuable products from microalgae include polyunsaturated fatty acids and pigments, clean and sustainable energy (e.g. biodiesel). Nevertheless, high cost for microalgae biomass harvesting has restricted the industrial application of microalgae. Flocculation, compared with other microalgae harvesting methods, has distinguished itself as a promising method with low cost and easy operation. Here, we reviewed the methods of microalgae harvesting using flocculation, including chemical flocculation, physical flocculation and biological flocculation, and the progress and prospect in bio-flocculation are especially focused. Harvesting microalgae via bio-flocculation, especially using bio-flocculant and microalgal strains that is self-flocculated, is one of the eco-friendly, cost-effective and efficient microalgae harvesting methods.
Biofuels ; Biomass ; Flocculation ; Microalgae ; growth & development

Ethanol tolerance is related to the expression of multiple genes, and genome-based engineering approaches are much more efficient than manipulation of single genes. In this study, ultraviolet (UV) mutagenesis, dielectric barrier discharge (DBD) air plasma mutagenesis, and artificial transcription factor (ATF) technology were adopted to treat an industrial yeast strain S. cerevisiae Sc4126 to obtain mutants with improved ethanol tolerance. Mutants with high ethanol tolerance were obtained, and the ratio of positive mutants was compared. Among the three approaches, the rate of positive mutation obtained by ATF technology was 10- to 100-folds of that of the two other methods, with highest genetic stability, suggesting the ATF technology promising for rapid alteration of phenotypes of industry yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; methods ; Mutagenesis ; Saccharomyces cerevisiae ; drug effects ; genetics ; growth & development

Chromosomal integration enables stable phenotype and therefore has become an important strategy for breeding of industrial Saccharomyces cerevisiae strains. pAUR135 is a plasmid that enables recycling use of antibiotic selection marker, and once attached with designated homologous sequences, integration vector for stable expression can be constructed. Development of S. cerevisiae strains by metabolic engineering normally demands overexpression of multiple genes, and employing pAUR135 plasmid, it is possible to construct S. cerevisiae strains by combinational integration of multiple genes in multiple sites, which results in different ratios of expressions of these genes. Xylose utilization pathway was taken as an example, with three pAUR135-based plasmids carrying three xylose assimilation genes constructed in this study. The three genes were sequentially integrated on the chromosome of S. cerevisiae by combinational integration. Xylose utilization rate was improved 24.4%-35.5% in the combinational integration strain comparing with that of the control strain with all the three genes integrated in one location. Strain improvement achieved by combinational integration is a novel method to manipulate multiple genes for genetic engineering of S. cerevisiae, and the recombinant strains are free of foreign sequences and selection markers. In addition, stable phenotype can be maintained, which is important for breeding of industrial strains. Therefore, combinational integration employing pAUR135 is a novel method for metabolic engineering of industrial S. cerevisiae strains.
Genetic Engineering ; methods ; Genetic Vectors ; Metabolic Engineering ; Plasmids ; genetics ; Saccharomyces cerevisiae ; genetics ; Xylose ; metabolism

Biofuels are renewable and environmentally friendly, but high production cost makes them economically not competitive, and the development of robust strains is thus one of the prerequisites. In this article, strain improvement studies based on the information from systems biology studies are reviewed, with a focus on their applications on stress tolerance improvement. Furthermore, the contribution of systems biology, synthetic biology and metabolic engineering in strain development for biofuel production is discussed, with an expectation for developing more robust strains for biofuel production.
Biofuels ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; trends ; Lignin ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; physiology ; Synthetic Biology ; methods ; Systems Biology ; methods

Biofuels and bioenergy not only benefit independence of energy supply, but also mitigate CO2 emissions. This special issue includes review reports and research articles involving various biofuels and bioenergy products and systems such as fuel ethanol, biodiesel, biogas, biohydrogen, microbial fuel cells and microbial electrolysis cells. Both fundamental research and technology development are highlighted. And in the meantime, challenges for large scale production and application of biofuels and bioenergy are discussed. Taking advantages of modern biotechnology advances, solutions to address these challenges are envisioned.
Bioelectric Energy Sources ; Biofuels ; Biotechnology ; trends ; Conservation of Energy Resources

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
Amino Acid Sequence ; Catalysis ; Coloring Agents ; metabolism ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peroxidases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Zymomonas ; enzymology

Improvement of stress tolerance to various adverse environmental conditions (such as toxic products, high temperature) of the industrial microorganisms is important for industrial applications. Ethanol produced by yeast fermentation is inhibitory to both yeast cell growth and metabolisms, and consequently is one of the key stress elements of brewer's yeast. Research on the biochemical and molecular mechanism of the tolerance of yeast can provide basis for breeding of yeast strain with improved ethanol tolerance. In recent years, employing global gene transcriptional analysis and functional analysis, new knowledge on the biochemical and molecular mechanisms of yeast ethanol tolerance has been accumulated, and novel genes and biochemical parameters related to ethanol tolerance have been revealed. Based on these studies, the overexpression and/or disruption of the related genes have successfully resulted in the breeding of new yeast strains with improved ethanol tolerance. This paper reviewed the recent research progress on the molecular mechanism of yeast ethanol tolerance, as well as the genetic engineering manipulations to improve yeast ethanol tolerance. The studies reviewed here not only deepened our knowledge on yeast ethanol tolerance, but also provided basis for more efficient bioconversion for bio-energy production.
Drug Tolerance ; genetics ; Ethanol ; metabolism ; pharmacology ; Fermentation ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; Saccharomyces cerevisiae ; drug effects ; genetics ; Saccharomyces cerevisiae Proteins ; genetics