Draft genome sequence of thermotolerant Saccharomyces cerevisiae AH465, isolated from Mt. Tatsuda, Kumamoto, Japan.

Thermotolerance is a required characteristic for biofuel production by yeast. Here, we report the draft genome sequence of thermotolerant Saccharomyces cerevisiae AH465, which was originally isolated by the authors. A hybrid assembly approach using MinION Mk1b and MiSeq sequencers was conducted. The assembled sequence comprises 13.4 Mb in 26 contigs.

Spathaspora quercus f.a., sp. nov., a novel yeast isolated from rotting wood and a lichen sample in Kyushu region, Japan.

Four novel d-xylose assimilation yeast strains were isolated from rotting wood and a lichen sample collected in the Kyushu region of Japan. Species identifications were performed by analysing the internal transcribed spacer 5.8S region sequences and the D1/D2 variable domain of the large subunit rRNA gene. Phylogenetic analysis suggested that these isolates are closely related to Spathaspora species isolated in China, such as S. jiuxiensis and S. parajiuxiensis. These isolates also showed sequence similarity to deposited sequences labelled as Schwanniomyces. They did not produce asci and ascospores under any of the test conditions. Based on phylogenetic analysis and phenotypic differences, Spathaspora quercus f.a., sp. nov. is proposed to accommodate these isolates. The holotype of Spathaspora quercus f.a., sp. nov. is NBRC 116146T (CBS18366). This species is able to ferment d-xylose, and a d-xylose fermentation test revealed that this species produces a considerable amount of xylitol.

Systematic approach for assessing whether undeletable chromosomal regions in Saccharomyces cerevisiae are required for cell viability.

Previously, we identified 49 undeletable chromosomal regions harboring only non-essential genes in the genome of Saccharomyces cerevisiae. We proposed that there might be unknown synthetic lethal combinations of genes present in such undeletable regions of the genome. In this study, we chose four of the smallest undeletable chromosomal regions among the 49 and performed extensive further analyses to narrow down the gene-pairs responsible for lethality by replacing sub-regions in various combinations with a DNA module comprising the CgLEU2 marker. Although the methodology was different from previous study, interestingly the results revealed that not only the sub-regions but also the entire region was replaceable. To solve the apparent discrepancy between previous and present results, we further conducted additional analysis including investigation of suppressor mutation and mini-chromosome loss assay through the construction of mini-chromosome harboring two particular chromosomal regions with marked with URA3 marker by employing 5-FOA system. Based upon careful observation on the phenotype of colony formation on 5-FOA medium by spot test, we came to an important conclusion that particular chromosomal regions harboring only non-essential genes can be categorized into three classes, i.e., essential, non-essential and intrinsically essential. Intrinsically essential region is defined as appearance of papillae after mini-chromosome loss which implicates that the region is essential but compensatable against cell lethality. Our present study indicates that prudent and multiple approaches as performed in this study are needed to judge whether a particular chromosomal region of the S. cerevisiae genome is essential, non-essential or intrinsically essential but compensatable.

CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae.

In our previous study, a novel genome engineering technology, PCR-mediated chromosome duplication (PCDup), was developed in Saccharomyces cerevisiae that enabled the duplication of any desired chromosomal region, resulting in a segmental aneuploid. From one round of transformation, PCDup can duplicate a single chromosomal region efficiently. However, simultaneous duplication of multiple chromosomal regions is not possible using PCDup technology, which is a serious drawback. Sequential duplication is possible, but this approach requires significantly more time and effort. Because PCDup depends upon homologous recombination, we reasoned that it might be possible to simultaneously create duplications of multiple chromosomal regions if we could increase the frequency of these events. Double-strand breaks have been shown to increase the frequency of homologous recombination around the break point. Thus, we aimed to integrate the genome editing tool CRISPR/Cas9 system, which induces double-strand breaks, with our conventional PCDup. The new method, which we named CRISPR-PCDup increased the efficiency of a single duplication by up to 30 fold. CRISPR-PCDup enabled the simultaneous duplication of long chromosomal segments (160 kb and 200 kb regions). Moreover, we were also able to increase the length of the duplicated chromosome by up to at least 400 kb, whereas conventional PCDup can duplicate up to a maximum of 300 kb. Given the enhanced efficiency of chromosomal segmental duplication and the saving in both labor and time, we propose that CRISPR-PCDup will be an invaluable technology for generating novel yeast strains with desirable traits for specific industrial applications and for investigating genome function in segmental aneuploid.

CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae.

Genome manipulation, especially the deletion or replacement of chromosomal regions, is a salient tool for the analysis of genome function. Because of low homologous recombination activity, however, current methods are limited to manipulating only one chromosomal region in a single transformation, making the simultaneous deletion or replacement of multiple chromosomal regions difficult, laborious, and time-consuming. Here, we have developed two highly efficient and versatile genome engineering technologies, named clustered regularly interspaced short palindromic repeats (CRISPR)-PCR-mediated chromosomal deletion (PCD) (CRISPR-PCD) and PCR-mediated chromosomal replacement (CRISPR-PCRep), that integrate the CRISPR-associated protein 9 (Cas9) genome editing system (CRISPR/Cas9) into, respectively, the PCD method for chromosomal deletion and our newly developed PCRep method for chromosomal replacement. Integration of CRISPR induces double strand breaks to activate homologous recombination, and thus enhances the efficiency of deletion by PCD and replacement by PCRep, enabling multiple chromosomal regions to be manipulated simultaneously for the first time. Our data show that CRISPR-PCD can delete two internal or terminal chromosomal regions, while CRISPR-PCRep can replace triple chromosomal regions simultaneously in a single transformation. Colony PCR analysis of structural alterations showed that triple replacement of four different sets of chromosomal regions was successful in 83%-100% of transformants analyzed. These novel genome engineering technologies, which greatly reduce time and labor for genome manipulation, will provide powerful tools to facilitate the simultaneous multiple deletion and replacement of chromosomal regions, enabling the rapid analysis of genome function and breeding of useful industrial yeast strains.

gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.

Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae.

To clarify the relationship between NAD(P)+/NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C. The TDH3p-GAL2 or gal80Δ strain completely consumed the xylose within 24 h and aerobically consumed 92-100% of the xylitol within 96 h, but anaerobically consumed only 20% of the xylitol within 96 h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD+ generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD+-dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD+ generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25 g/L, 5.30 g/L, and 4.27 g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward.

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions.

Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.

Development of industrial yeast strain with improved acid- and thermo-tolerance through evolution under continuous fermentation conditions followed by haploidization and mating.

Continuous fermentation using the industrial Saccharomyces cerevisiae diploid strain WW was carried out under acidic or high-temperature conditions to achieve acid- or thermo-tolerant mutants. Mutants isolated at pH 2.5 and 41°C showed improved growth and fermentation ability under acidic and elevated temperature conditions. Haploid strains WW17A1 and WW17A4 obtained from the mutated diploid strain WW17A showed better growth and 4.5-6.5% higher ethanol yields at pH 2.7 than the original strains. Haploid strain WW12T4 obtained from mutated diploid strain WW12T showed 1.25-1.50 times and 2.8-4.7 times higher total cell number and cell viability, respectively, than the original strains at 42°C. Strain AT, which had significantly improved acid- and thermo-tolerance, was developed by mating strain WW17A1 with WW12T4. Batch fermentation at 41°C and pH 3.5 showed that the ethanol concentration and yield achieved during fermentation by strain AT were 55.4 g/L and 72.5%, respectively, which were 10 g/L and 13.4% higher than that of the original strain WW. The present study demonstrates that continuous cultivation followed by haploidization and mating is a powerful approach for enhancing the tolerance of industrial strains.

Draft Genome Sequence of Saccharomyces cerevisiae NAM34-4C, a Lactic Acid-Assimilating Industrial Yeast Strain.

We determined the genome sequence of industrial Saccharomyces cerevisiae strain NAM34-4C, which would be useful for bioethanol production. The approximately 11.5-Mb draft genome sequence of NAM34-4C will provide remarkable insights into metabolic engineering for effective production of bioethanol from biomass.

Potent L-lactic acid assimilation of the fermentative and heterothallic haploid yeast Saccharomyces cerevisiae NAM34-4C.

We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploid strains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies. The first strategy was as follows: (i) the KF7-5C was crossed with the laboratory strain SH6710; (ii) the progenies were backcrossed with KF7-5C three times; and (iii) the progenies were inbred three times to maintain a genetic background close to that of KF7. The NAM12 diploid between the cross of the resultant two strains, NAM11-9C and NAM11-13A, showed efficient sporulation and exhibited excellent growth in YPD medium (pH 3.5) at 35°C with 1.4-h generation time, indicating thermotolerance and acid tolerance. The second strategy was successive intrastrain crosses. The resultant two strains, KFG4-6B and KFG4-4B, showed excellent mating capacity. A spontaneous mutant of KFG4-6B, KFG4-6BD, showed a high growth rate with a generation time of 1.1 h in YPD medium (pH 3.0) at 35°C. The KFG4-6BD strain produced ascospores, which were crossed with NAM11-2C and its progeny to produce tetrads. These tetrads were crossed with KFG4-4B to produce NAM26-14A and NAM26-15A. The latter strain had a generation time of 1.6 h at 35°C in pH 2.5, thus exhibiting further thermotolerance and acid tolerance. A progeny from a cross of NAM26-14A and NAM26-15A yielded the strain NAM34-4C, which showed potent lactic acid assimilation and high transformation efficiency, better than those of a standard laboratory strain.

Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization.

A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h.

Improvement of ethanol production from D-lactic acid by constitutive expression of lactate transporter Jen1p in Saccharomyces cerevisiae.

To improve ethanol production from D-lactate, Jen1p, a monocarboxylate-proton symporter, was constitutively expressed in Saccharomyces cerevisiae NAM34-4C. The mutant produced 2.4 g/L of ethanol, approximately 2.4 times higher than that of the wild-type strain. A monocarboxylate/proton symporter gene (JEN1) null mutant was also constructed. It produced 0.19 g/L of ethanol, 5 times lower than that of the wild-type strain.

Ethanol production from D-lactic acid by lactic acid-assimilating Saccharomyces cerevisiae NAM34-4C.

The lactic acid-assimilating yeast Saccharomyces cerevisiae NAM34-4C grew rapidly in minimal D-lactate medium (pH 3.5) at 35°C, compared with minimal L-lactate medium. A laboratory strain, S. cerevisiae S288C, did not grow in either medium at pH 3.5. Strain NAM34-4C produced remarkably high levels of ethanol in YPDL medium at pH 3.5, but not at pH 5.5, when D-lactate was provided as the carbon source. Optimal cultivation conditions for ethanol production from D-lactate by strain NAM34-4C were as follows: shaking speed, 60 rpm; initial pH, 3.0; cultivation temperature, 35°C; yeast extract, 5 g/L; peptone, 10 g/L; and D-lactate, 30 g/L. Under these conditions, strain NAM34-4C produced 2.7 g/L ethanol, which is 18% of the theoretical maximal yield (0.51 3 initial D-lactate concentration).

A simple assay for 6-aminohexanoate-oligomer-hydrolase using N-(4-nitrophenyl)-6-aminohexanamide.

We synthesized N-(4-nitrophenyl)-6-aminohexanamide (AHpNA) and used it as a substrate in a kinetic study of 6-aminohexanoate-hydrolase (NylB), a nylon oligomer-hydrolyzing enzyme. NylBs derived from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 hydrolyzed AHpNA as well as a 6-aminohexanoic acid dimer, a known substrate for NylB. The K(m) values of the NylB from Arthrobacter sp. KI72 and Pseudomonas sp. NK87 for AHpNA were 0.5 mM and 2.0 mM, respectively.

Plasmid transformation of competent Bacillus subtilis by lysed protoplast DNA.

Transformation of competent Bacillus subtilis with DNA obtained from lysed protoplasts (LP transformation) was analyzed using several different plasmid vectors: pC194, pUB110, pCB1 (consisting of pC194 and pBluescript II SK+), and pAC32R2 (consisting of pUB110 derivative and pUC19). LP transformation of B. subtilis QB936 with pCB1 was 6500-fold higher than that achieved using conventional transformation with purified DNA. Greater transformation efficiencies were also obtained using pAC32R2. However, transformation frequencies using both protoplast-derived and purified pC194 were very low (1.4-2.0×10(2) transformants per µg DNA). Hence, the efficiency of transformation depends on the nucleotide sequence of the donor plasmid. The LP transformation frequency using pC194 obtained from an add5 mutant was remarkably enhanced (1.6×10(8) transformants per µg DNA), indicating that this unique form of high molecular weight DNA is likely responsible for part of the stimulatory effect. Chromosomal DNA inhibited plasmid transformation using pC194 and pUB110, but had little effect on pCB1 transformation. Conversely, pCB1 DNA did not inhibit transformation with protoplast-derived chromosomal DNA. Competence proteins under the control of transcription factor ComK were likely required for LP plasmid transformation. The DNA concentration-dependence of plasmid transformation was first order and the slope value was one.

Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis.

The role of the competence protein ComEA in DNA uptake during transformation of competent Bacillus subtilis was analyzed by lysed-protoplast transformation (LP transformation). A comEA deletion mutant was constructed by a fusion polymerase chain reaction. Transformants of the mutant were obtained by LP transformation at a frequency of 1.1 × 10(2) transformants per µg DNA, representing a low relative efficiency of transformation [RET (mutant/wild type)] of 2.7 × 10(-6). This implied an important role of the protein during DNA uptake. When analyzing LP transformation of comEA with a plasmid (5.7 kb), a similar RET (mutant/wild type) of 5.6 × 10(-5) was obtained. Following addition of DNA into the comEA mutant culture, the number of transformants increased at a rate of 0.5 transformants/min, which was very low compared with the wild-type (6.9×10(4) transformants/min). However, even in the comEA mutant, DNA uptake began immediately after addition of DNA. Using co-transformation analysis of the comEA mutant, short linkages at distances of 2-156 kb could be detected, but not long linkages at distances of 671-1662 kb. Taken together, the results indicate that ComEA plays an important role in the transfer of transforming DNA into the DNA channel and in controlling the rate of DNA uptake.

Essential involvement of the Bacillus subtilis ABC transporter, EcsB, in genetic transformation of purified DNA but not native DNA from protoplast lysates.

Involvement of the Bacillus subtilis ABC transporter EcsB in genetic transformation with native DNA from protoplast lysate (LP transformation) was investigated using an ecsB deletion mutant constructed by fusion polymerase chain reaction. In these experiments, the non-transformability phenotype of the ecsB mutant was reversed and high numbers of transformants generated (1.5×10(5)/µg DNA). The relative efficiency of transformation (RET) of ecsB to wild type (1.2×10(-2)) was a thousand times higher using native chromosomal DNA than the RET obtained from purified DNA (<8.6×10(-6)). Similar transformation efficiencies were observed using native plasmid DNA. These results rule out a primary role for EcsB as a competence gene regulator. DNA-binding proteins attached to native DNA are not present in purified DNA preparations, and it is possible that such proteins could account for the transformability of the ecsB mutant. Because EcsB may play a role in protein(s) export, we tested exogenous proteins to identify functional replacements. We found that bovine serum albumin (fraction V) partially suppressed the phenotype of the ecsB mutation, leading to transformability with purified DNA. Linkage analysis of the ecsB mutant by LP co-transformation produced a higher co-transformation ratio (42% and 20%) at a distance of 34kb and 121kb in the ecsB mutant, compared to the wild-type strain, AYG2 (30.5% and 12.3%). The stimulatory linkage effect observed could be derived from a regulating gene involved in homologous recombination.