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

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

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

BACKGROUND:Artificial femoral head replacement provides a new idea for the repair of unstable intertrochanteric fracture. Artificial prosthesis replacement may affect original femoral biomechanical stability and lead to a variety of adverse consequences. OBJECTIVE:To analyze the stress distribution of femoral head replacement in the treatment of unstable femoral intertrochanteric fractures with three-dimensional finite element analysis. METHODS:One male old volunteer was randomly selected from population who underwent health examination. The left femur was scanned with spiral CT, and the three-dimensional finite element models of the human femur and prosthesis were established. The three-dimensional finite element model was used to simulate the actual working conditions of human climbing stairs, and the stress distribution of the bone channels around the surface of the femur and the prosthesis was analyzed with three-dimensional finite element analysis. RESULTS AND CONCLUSION: Under normal condition, the stress of the human femur was in a consistent state. Stress changed gradualy from the proximal end to the distal end. The stress of the prosthesis was concentrated in the middle section. The prosthesis of inner stress distribution was analyzed to obtain stress distribution of prosthesis and femur cancelous bone interface. The analysis found that stress change trend was consistent. The results suggest that artificial femoral head replacement does not have a significant effect on the overal stress distribution of the human femur, and the overal stress distribution does not change, and the maximum stress region is located in the middle of the whole femur. After the reconstruction, the stress concentration of the femur is not observed.

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

Ethanol tolerance of self-flocculating yeast SPSC01 was studied in a 3-L bioreactor under fed-batch culture. Yeast floc populations with the average sizes around 100, 200, 300, and 400 microm were obtained by adjusting the mechanical stirring rates of the fermentation system. When subjected to 20% (V/V) ethanol shock for 6 h at 30 degrees C, the remained cell viability was 3.5%, 26.7%, 48.8% and 37.6% for the aforementioned four floc populations, respectively. The highest ethanol yield 85.5% was achieved for the 300 microm flocs, 7.2% higher than that of the 100 microm flocs. The amounts of trehalose and ergosterol (including free ergosterol and total ergosterol) were positively correlated with the average size distributions from 100 to 300 microm. However, in the 400 microm flocs, the content of trehalose and ergosterol decreased, which coincided with its reduced ethanol tolerance compared to that of the 300 microm flocs. Furthermore, when subjected to 15% (V/V) ethanol shock at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient(P') of the 300 microm flocs accounted for only 43% and 52% respectively of those of the 100 microm and 200 microm populations. The effect of floc size distribution on the ethanol tolerance of the self-flocculating yeast strain SPSC01 was closely related to plasma membrane permeability. An optimal floc size distribution with the highest ethanol tolerance and ethanol production level could be obtained by controlling mechanical stirring speed of the bioreactor, which provides basis for the process optimization of fuel ethanol production using this self-flocculating strain.
Bioreactors ; microbiology ; Drug Tolerance ; Ergosterol ; biosynthesis ; Ethanol ; metabolism ; pharmacology ; Fermentation ; Flocculation ; Industrial Microbiology ; methods ; Particle Size ; Trehalose ; biosynthesis ; Yeasts ; drug effects ; growth & development ; metabolism

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

Directed evolution of transcription factors can be employed to effectively improve the phenotypes which are controlled by multiple genetic loci. In this study, we used error-prone PCR for the directed evolution of SPT3, which is the component of yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex responsible for the transcription of stress-related genes, and studied its effect on the improvement of ethanol tolerance. Mutant library was constructed by ligating the error-prone PCR products with a modified pYES2.0 plasmid, and the expression plasmids were subsequently transformed to yeast industrial strain Saccharomyces cerevisiae 4126. One mutant strain M25 showing superior growth in presence of 10% ethanol was selected. M25 produced 11.7% more ethanol than the control strain harboring the empty vector when 125 g/L glucose was used as substrate. This study revealed that SPT3 is an important transcription factor for the metabolic engineering of yeast ethanol tolerance.
Directed Molecular Evolution ; methods ; Drug Resistance, Fungal ; Drug Tolerance ; Ethanol ; metabolism ; pharmacology ; Industrial Microbiology ; methods ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; Trans-Activators ; genetics ; Transcription Factors ; genetics