Pin-Mei Wang

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.

A Novel Strategy to Construct Yeast Saccharomyces cerevisiae Strains for Very High Gravity Fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.

Optimization and characterization of polysaccharide-based bioflocculant produced by Paenibacillus elgii B69 and its application in wastewater treatment

The optimization, purification and characterization of bioflocculant produced by Paenibacillus elgii B69 were investigated. The bioflocculant was an exopolysaccharide composed of glucose, glucuronic acid, mannose and xylose. The maximum bioflocculant production was about 25.63 g/L achieved with sucrose at 51.35 g/L, peptone at 6.78 g/L and yeast extract at 0.47 g/L optimized by response-surface methodology. In addition, a series of experiments was performed to investigate the flocculation activities towards kaolin clay, dyeing pigment, heavy metal ion, and real wastewater and the result indicated the new bioflocculant had high activities towards all the tested pollutions. These results showed its great potential for water pretreatment used in industry.

Genome sequencing and genetic breeding of a bioethanol Saccharomyces cerevisiae strain YJS329

BackgroundEnvironmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits.ResultsWe explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified.ConclusionsThrough comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.

Relationship of trehalose accumulation with ethanol fermentation in industrial Saccharomyces cerevisiae yeast strains

The protective effect and the mechanisms of trehalose accumulation in industrial Saccharomyces cerevisiae strains were investigated during ethanol fermentation. The engineered strains with more intercellular trehalose achieved significantly higher fermentation rates and ethanol yields than their wild strain ZS during very high gravity (VHG) fermentation, while their performances were not different during regular fermentation. The VHG fermentation performances of these strains were consistent with their growth capacity under osmotic stress and ethanol stress, the key stress factors during VHG fermentation. These results suggest that trehalose accumulation is more important for VHG fermentation of industrial yeast strains than regular one. The differences in membrane integrity and antioxidative capacity of these strains indicated the possible mechanisms of trehalose as a protectant under VHG condition. Therefore, trehalose metabolic engineering may be a useful strategy for improving the VHG fermentation performance of industrial yeast strains.

Comparative functional genomics to reveal the molecular basis of phenotypic diversities and guide the genetic breeding of industrial yeast strains.

An understanding of the genetic basis underlying the phenotypic variations of yeast strains would guide the breeding of this useful microorganism. Here, comparative functional genomics (CFG) of two bioethanol Saccharomyces cerevisiae strains (YJS329 and ZK2) with different stress tolerances and ethanol fermentation performances were performed. Our analysis indicated that different patterns of gene expression in the central carbon metabolism, antioxidative factors, and membrane compositions of these two strains are the main contributors to their various traits. Some of the differently expressed genes were directly caused by the genomic structural variations between YJS329 and ZK2. Moreover, CFG of these two strains also led to novel insights into the mechanism of stress tolerance in yeast. For example, it was found that more oleic acid in the plasma membrane contributes to the acetic acid tolerance of yeast. Based on the genetic information particular to each strain, strategies to improve their adaptability and ethanol fermentation performances were designed and confirmed. Thus, CFG could not only help reveal basis of phenotypic diversities but also guide the genetic breeding of industrial microorganisms.

The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae.

A challenge associated with the ethanol productivity under very-high-gravity (VHG) conditions, optimizing multi-traits (i.e. byproduct formation and stress tolerance) of industrial yeast strains, is overcome by a combination of metabolic engineering and genome shuffling. First, industrial strain Y12 was deleted with a glycerol exporter Fps1p and hetero-expressed with glyceraldehydes-3-phosphate dehydrogenase, resulting in the modified strain YFG12 with lower glycerol yield. Second, YFG12 was subjected to three rounds of drug resistance marker-aided genome shuffling to increase its ethanol tolerance, and the best shuffled strain TS5 was obtained. Compared with wild strain Y12, shuffled strain TS5 not only decreased glycerol formation by 14.8%, but also increased fermentation rate and ethanol yield by 3.7% and 7.6%, respectively. Moreover, the system of genetic modification and Cre/loxP in aid of three different drug-resistance markers presented in the study significantly improved breeding efficiency and will facilitate the application of breeding technologies in prototrophic industrial microorganisms.

Genomic structural variations contribute to trait improvement during whole-genome shuffling of yeast

Whole-genome shuffling (WGS) is a powerful technology of improving the complex traits of many microorganisms. However, the molecular mechanisms underlying the altered phenotypes in isolates were less clarified. Isolates with significantly enhanced stress tolerance and ethanol titer under very-high-gravity conditions were obtained after WGS of the bioethanol Saccharomyces cerevisiae strain ZTW1. Karyotype analysis and RT-qPCR showed that chromosomal rearrangement occurred frequently in genome shuffling. Thus, the phenotypic effects of genomic structural variations were determined in this study. RNA-Seq and physiological analyses revealed the diverse transcription pattern and physiological status of the isolate S3-110 and ZTW1. Our observations suggest that the improved stress tolerance of S3-110 can be largely attributed to the copy number variations in large DNA regions, which would adjust the ploidy of yeast cells and expression levels of certain genes involved in stress response. Overall, this work not only constructed shuffled S. cerevisiae strains that have potential industrial applications but also provided novel insights into the molecular mechanisms of WGS and enhanced our knowledge on this useful breeding strategy.

Increasing viscosity and yields of bacterial exopolysaccharides by repeatedly exposing strains to ampicillin

A universal method to enhance productivity and viscosity of bacterial exopolysaccharides was developed. The technique was based on the principle that ampicillin can inhibit the biosynthesis of peptidoglycan, which shares a common synthetic pathway with that of bacterial exopolysaccharides. Serial passages of three typical representatives of bacterial EPS-producing strains, namely Sphingomonas elodea, Xanthomonas campestris, and Paenibacillus elgii, were subjected to ampicillin, which was used as a stressor and a mutagen. These mutant strains are advantageous over other strains because of two major factors. First, all of the resulting strains were almost mutants with increase in EPS productivity and viscosity. Second, isolated serial strains showed different levels of increase in EPS production and viscosity to satisfy the different requirements of practical applications. No differences were observed in the monosaccharide composition produced by the mutant and parent strains; however, high-viscosity mutant strains exhibited higher molecular weights. The results confirmed that the developed method is a controlled universal one that can improve exopolysaccharides productivity and viscosity.

Two UDP-glucuronic acid decarboxylases involved in the biosynthesis of a bacterial exopolysaccharide in Paenibacillus elgii

Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.