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Two yellow-pigmented, Gram-stain-negative, aerobic, rod-shaped bacteria were isolated from the water of the hypersaline Chaka Salt Lake (strain SaA2.12T) and sediment of Qinghai Lake (strain LaA7.5T), PR China. According to the 16S rRNA phylogeny, the isolates belong to the genus Flavobacterium , showing the highest 16S rRNA sequence similarities to Flavobacterium arcticum SM1502T(97.6–97.7 %) and Flavobacterium suzhouense XIN-1T(96.5–96.6 %). Moreover, strains SaA2.12T and LaA7.5T showed 99.73 % 16S rRNA sequence similarity to each other. Major fatty acids, respiratory quinones and polar lipids detected in these isolates were iso-C15 : 0, menaquinone-6 and phosphatidylethanolamine, respectively. Strains SaA2.12T and LaA7.5T showed significant unique characteristics between them as well as between the closest phylogenetic members. The highest digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between SaA2.12T and its closest neighbours were 25.3 and 82.8 %, respectively; whereas these values (highest) between LaA7.5T and its closest members were 25.2 and 82.8 %, respectively. The dDDH and ANI values between strains SaA2.12T and LaA7.5T were calculated as 75.9 and 97.2 %, respectively. Therefore, based on polyphasic data, we propose that strain SaA2.12T represents a novel species with the name Flavobacterium salilacus sp. nov., with the type strain SaA2.12T (=KCTC 72220T=MCCC 1K03618T) and strain LaA7.5T as a subspecies within novel Flavobacterium salilacus with the name Flavobacterium salilacus subsp. altitudinum subsp. nov., with the type strain LaA7.5T (=KCTC 72806T=MCCC 1K04372T). These propositions automatically create Flavobacterium salilacus subsp. salilacus subsp. nov. with SaA2.12T (=KCTC 72220T=MCCC 1K03618T) as the type strain.

Chromosomal rearrangements comprise unbalanced structural variations resulting in gain or loss of DNA copy numbers, as well as balanced events including translocation and inversion that are copy number neutral, both of which contribute to phenotypic evolution in organisms. The exquisite genetic assay and gene editing tools available for the model organism Saccharomyces cerevisiae facilitate deep exploration of the mechanisms underlying chromosomal rearrangements. We discuss here the pathways and influential factors of chromosomal rearrangements in S. cerevisiae. Several methods have been developed to generate on-demand chromosomal rearrangements and map the breakpoints of rearrangement events. Finally, we highlight the contributions of chromosomal rearrangements to drive phenotypic evolution in various S. cerevisiae strains. Given the evolutionary conservation of DNA replication and recombination in organisms, the knowledge gathered in the small genome of yeast can be extended to the genomes of higher eukaryotes.

The aim of this work was to develop a novel strategy for improving the vanillin tolerance and ethanol fermentation performances of Saccharomyces cerevisiae strains. Isogeneic diploid, triploid, and tetraploid S. cerevisiae strains were generated by genome duplication of haploid strain CEN.PK2-1C. Ploidy increments improved vanillin tolerance and diminished proliferation capability. Antimitotic drug methyl benzimidazol-2-ylcarbamate (MBC) was used to introduce chromosomal aberrations into the tetraploid S. cerevisiae strain. Interestingly, aneuploid mutants with DNA contents between triploid and tetraploid were more resistant to vanillin and showed faster ethanol fermentation rates than all euploid strains. The physiological characteristics of these mutants suggest that higher bioconversion capacities of vanillin and ergosterol contents might contribute to improved vanillin tolerance. This study demonstrates that genome duplication and MBC treatment is a powerful strategy to improve the vanillin tolerance of yeast strains.

A novel bacterium designated SSM4.2T was isolated from seaweed of Gouqi Island, which is the center of the Zhoushan fishing ground in the East China Sea. Strain SSM4.2T was Gram-stain-negative, bright yellow-pigmented, short rod-shaped, non-flagellated, non-spore forming, aerobic and motile by gliding. Growth was observed at 4–37 °C (optimum 25–30 °C), pH 6.0–8.0 (optimum pH 7.0) and 0–2.0% (w/v) NaCl (optimum 0%) concentration. The strain was catalase- and oxidase-positive. Menaquinone-6 (MK-6) was found as the sole respiratory quinone and zeaxanthin as the main carotenoid pigment. The predominant fatty acids (≥ 10%) were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1 ω7c /C16:1 ω6c). The major polar lipid was phosphatidylethanolamine (PE). The genome size was 5.7 Mbp. The DNA G + C content was 34.1 mol%. 16S rRNA gene sequence revealed that strain SSM4.2T belongs to the genus Flavobacterium and shares high-sequence similarity with F. limi KACC 18851T (98.1%), F. hydrophilum KACC 19591T (97.6%), F. defluvii KCTC 12612T (97.1%), F. cheongpyeongense KACC 19592T (97.0%) and F. fluviatile KCTC 52446T (96.9%). Strain SSM4.2T had 73.2–84.6% average nucleotide identity and 19.1–29.4% digital DNA-DNA hybridization values with its closest type strains. Based on its phenotypic, chemotaxonomic, phylogenetic and genomic features, strain SSM4.2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium ajazii sp. nov. is proposed. The type strain is SSM4.2T (= KCTC 72807T = MCCC 1K04370T).

A yellow pigmented, Gram-stain-negative, aerobic bacterium designated A5.7T was studied to evaluate the taxonomic position following the modern polyphasic approach. The strain was isolated from sediments near Zhairuo Island, which is situated in the East China Sea. Cells were non-spore forming rods without flagella but showed motility by gliding. Growth was observed at 15–35°C (optimum 28°C), pH 6.0–9.0 (optimum pH 6.5) and 0–2% (w/v) NaCl (optimum 0–0.5%) in LB broth. The major respiratory quinone of A5.7T was menaquinone 6. The major polar lipid of A5.7T was phosphatidylethanolamine and the predominant fatty acids (> 5%) were iso-C15:0, iso-C17:0 3-OH, C15:1ω6c, iso-C15:0 3-OH, iso-C15:1 G, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 9 (iso-C17:1ω9c and/or C16:010-methyl). Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belongs to the genus Flavobacterium and shares the highest sequence similarities with Flavobacterium sharifuzzamanii A7.6T (98.5%), Flavobacterium tistrianum GB 56.1T (98.3%), Flavobacterium nitrogenifigens NXU-44T (97.8%), Flavobacterium anhuiense D3T (97.6%), Flavobacterium ginsenosidimutans THG 01T (97.6%), and Flavobacterium foetidum CJ42T (97.6%). Digital DNA-DNA hybridization and average nucleotide identity values between the strain and its closest phylogenetic neighbors showed the ranges from 19.6 to 34.1% and 73.7 to 87.9%, respectively. Therefore, based on polyphasic characteristics, strain A5.7T represents a novel species of the genus Flavobacterium for which the name Flavobacterium zhairuonensis sp. nov. is proposed. The type strain is A5.7T (= KCTC 62406T = MCCC 1K03494T).

Oxidative stress has been implicated in a variety of human diseases. One plausible mechanism is that reactive active species can induce DNA damages and jeopardize genome integrity. To explore how oxidative stress results in global genomic instability in cells, our current study examined the genomic alterations caused by H2O2 exposure at the whole genome level in yeast. Using SNP microarrays and genome sequencing, we mapped H2O2-induced genomic alterations in the yeast genome ranging from point mutations and mitotic recombination to chromosomal aneuploidy. Our results suggested most H2O2-induced mitotic recombination events were the result of DNA double-stand breaks generated by hydroxyl radicals. Moreover, the mutagenic effect of H2O2 was shown to be largely dependent on DNA polymerase ζ. Lastly, we showed that H2O2 exposure allows rapid phenotypic evolution in yeast strains. Our findings indicate DNA lesions resulting from H2O2 may be general factors that drive genome instability and phenotypic evolution in organisms.

To improve tolerance to acetic acid that is present in lignocellulosic hydrolysates and affects bioethanol production by Saccharomyces cerevisiae. Saccharomyces cerevisiae strains with improved tolerance to acetic acid were obtained through deletion of the JJJ1 gene. The lag phase of the JJJ1 deletion mutant BYΔJJJ1 was ~16 h shorter than that of the parent strain, BY4741, when the fermentation medium contained 4.5 g acetic acid/l. Additionally, the specific ethanol production rate of BYΔJJJ1 was increased (0.057 g/g h) compared to that of the parent strain (0.051 g/g h). Comparative transcription and physiological analyses revealed higher long chain fatty acid, trehalose, and catalase contents might be critical factors responsible for the acetic acid resistance of JJJ1 knockout strains. JJJ1 deletion improves acetic acid tolerance and ethanol fermentation performance of S. cerevisiae.

The yeast Saccharomyces cerevisiae has been widely used as a model system for studying the physiological and pharmacological action of small-molecular drugs. Here, a heterozygous diploid S. cerevisiae strain QSS4 was generated to determine whether drugs could induce chromosomal instability by determining the frequency of mitotic recombination. Using the combination of a custom SNP microarray and yeast screening system, the patterns of chromosomal instability induced by drugs were explored at the whole genome level in QSS4. We found that Zeocin (a member of the bleomycin family) treatment increased the rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement over a hundred-fold. Most recombination events are likely to be initiated by DNA double-stand breaks directly generated by Zeocin. Another remarkable finding is that G4-motifs and low GC regions were significantly underrepresented within the gene conversion tracts of Zeocin-induced LOH events, indicating that certain DNA regions are less preferred Zeocin-binding sites in vivo. This study provides a novel paradigm for evaluating genetic toxicity of small-molecular drugs using yeast models.

A novel bacterial strain A7.6T was isolated from the sediments collected near the Zhairuo Island located in the East China Sea and characterized using a polyphasic approach. Cells were Gram-stain-negative, rod-shaped, non-spore forming, non-flagellated but motile by gliding. The strain was aerobic, positive for oxidase and catalase activities. The strain can grow at 4–35 °C, pH 5.5–9.0, and 0–3% (w/v) NaCl concentration. The major polar lipid was phosphatidylethanolamine, the predominant fatty acids (> 10%) were iso-C15:0 and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The genomic G+C content was 33.6 mol% and the major respiratory quinone was menaquinone 6. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A7.6T belonged to the genus Flavobacterium and was closely related to Flavobacterium tistrianum GB 56.1T (98.4% similarity), F. nitrogenifigens NXU-44T (98.4%), F. ginsenosidimutans THG 01T (98.0%) and F. anhuiense D3T (97.7%). Average nucleotide identities and digital DNA–DNA hybridizations values for genomes ranged from 75.9 to 91.4% and 21.4 to 43.9% between strain A7.6T and its closest phylogenetic neighbors. The polyphasic characterization indicated that strain A7.6T represented a novel species of the genus Flavobacterium, for which the name Flavobacterium sharifuzzamanii is proposed. The type strain is A7.6T (= KCTC 62405T = MCCC 1K03485T). The NCBI GenBank accession number for the 16S rRNA gene of A7.6T is MH396692, and for the genome sequence is QJGZ00000000. The digital protologue database (DPD) Taxon Number is TA00643.

Yeast Saccharomyces cerevisiae strains isolated from different sources generally show extensive genetic and phenotypic diversity. Understanding how genomic variations influence phenotypes is important for developing strategies with improved economic traits. The diploid S. cerevisiae strain NY1308 is used for cellulosic bioethanol production. Whole genome sequencing identified an extensive amount of single nucleotide variations and small insertions/deletions in the genome of NY1308 compared with the S288c genome. Gene annotation of the assembled NY1308 genome showed that 43 unique genes are absent in the S288c genome. Phylogenetic analysis suggested most of the unique genes were obtained through horizontal gene transfer from other species. RNA-Seq revealed that some unique genes were not functional in NY1308 due to unidentified intron sequences. During bioethanol fermentation, NY1308 tends to flocculate when certain inhibitors (derived from the pretreatment of cellulosic feedstock) are present in the fermentation medium. qRT-PCR and genetic manipulation confirmed that the novel gene, NYn43, contributed to the flocculation ability of NY1308. Deletion of NYn43 resulted in a faster fermentation rate for NY1308. This work disclosed the genetic characterization of a bioethanol-producing S. cerevisiae strain and provided a useful paradigm showing how the genetic diversity of the yeast population would facilitate the personalized development of desirable traits.

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.

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.

Polyploidy is common in Saccharomyces cerevisiae strains, but the physiological and phenotypic effects of ploidy changes have not been fully clarified. Here, isogenic diploid, triploid, and tetraploid S. cerevisiae strains were constructed from a haploid strain, CEN.PK2-1C. Stress tolerance and ethanol fermentation performance of the four euploid strains were compared. Each euploid strain had strengths and weaknesses in tolerance to certain stressors, and no single strain was tolerant of all stressors. The diploid had higher ethanol production than the other strains in normal fermentation medium, while the triploid strain showed the fastest fermentation rate in the presence of inhibitors found in lignocellulosic hydrolysate. Physiological determination revealed diverse physiological attributes, such as trehalose, ergosterol, glutathione, and anti-oxidative enzymes among the strains. Our analyses suggest that both ploidy parity and number of chromosome sets contribute to changes in physiological status. Using qRT-PCR, different expression patterns of genes involved in the regulation of cell morphology and the biosynthesis of key physiological attributes among strains were determined. Our data provide novel insights into the multiple effects of genome duplication on yeast cells and are a useful reference for breeding excellent strains used in specific industrial applications.

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.

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.

To overcome the drawbacks of protoplast fusion in industrial breeding, strain-specific molecular markers were applied to select hybrids of industrial Saccharomyces cerevisiae strains. Random Amplified Polymorphic DNA (RAPD) analysis was used to generate strain-specific RAPD markers for two industrial yeast strains, Z8 and Z9. For industrial and technical controls, two RAPD markers with non-coding regions were converted into stable Sequence Characterized Amplified Region (SCAR) markers. Hybrids of Z8 and Z9 were obtained by protoplast fusion in combination with SCAR markers and were found to increase ethanol production by 4.3–8.1%. Results suggested that protoplast fusion could be combined with RAPD-SCAR molecular markers and applied in industrial breeding instead of auxotrophic markers.

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.

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.

Genomic structural variation (GSV) is a ubiquitous phenomenon observed in the genomes of Saccharomyces cerevisiae strains with different genetic backgrounds; however, the physiological and phenotypic effects of GSV are not well understood. Here, we first revealed the genetic characteristics of a widely used industrial S. cerevisiae strain, ZTW1, by whole genome sequencing. ZTW1 was identified as an aneuploidy strain and a large-scale GSV was observed in the ZTW1 genome compared with the genome of a diploid strain YJS329. These GSV events led to copy number variations (CNVs) in many chromosomal segments as well as one whole chromosome in the ZTW1 genome. Changes in the DNA dosage of certain functional genes directly affected their expression levels and the resultant ZTW1 phenotypes. Moreover, CNVs of large chromosomal regions triggered an aneuploidy stress in ZTW1. This stress decreased the proliferation ability and tolerance of ZTW1 to various stresses, while aneuploidy response stress may also provide some benefits to the fermentation performance of the yeast, including increased fermentation rates and decreased byproduct generation. This work reveals genomic characters of the bioethanol S. cerevisiae strain ZTW1 and suggests that GSV is an important kind of mutation that changes the traits of industrial S. cerevisiae strains.