Kentaro Inokuma

Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping

To improve isopropanol production by metabolically engineered Escherichia coli strain TA76, the optimization of fermentation conditions and isopropanol removal by gas stripping were performed. Isopropanol is one of the simplest secondary alcohols, and it can be dehydrated to yield propylene, which is currently derived from petroleum as a monomer for making polypropylene. Initially, using a pH-controlled fed-batch culture with the intermittent addition of glucose, strain TA76 produced 667 mM (40.1g/L) of isopropanol after 60 h, representing 73.2% (mol isopropanol/mol glucose) of the theoretical maximum yield. Because the accumulation of isopropanol drastically reduced production yields, a gas stripping recovery method was incorporated into the fed-batch culture system. Using this approach, strain TA76 produced 2378 mM (143 g/L) of isopropanol after 240 h with a yield of 67.4% (mol/mol). To our knowledge, this titer represents the highest level of isopropanol production by E. coli to date and suggests that strain TA76 has a great potential for commercial fermentative isopropanol production.

Efficient yeast cell-surface display of exo- and endo-cellulase using the SED1 anchoring region and its original promoter

BackgroundThe recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for direct conversion of lignocellulosic materials to ethanol. However, the cellulolytic activities of the conventional cellulase-displaying yeast strains are insufficient for the hydrolysis of cellulose. In this study, we constructed novel gene cassettes for the efficient cellulose utilization by cellulase-displaying yeast strains.ResultsThe novel gene cassettes for the cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reeseii endoglucanase II (EGII) were constructed using the promoter and the GPI anchoring region derived from Saccharomyces cerevisiae SED1. The gene cassettes were integrated into the S. cerevisiae genome, then the β-glucosidase activity of these recombinant strains was evaluated. We revealed that simultaneous utilization of the SED1 promoter and Sed1 anchoring domain in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The β-glucosidase activity of recombinant yeast cells transduced with the novel gene cassette was 8.4-fold higher than that of a conventional strain. The novel EGII-displaying strain also achieved 106-fold higher hydrolysis activity against the water-insoluble cellulose than a conventional strain. Furthermore, direct ethanol production from hydrothermally processed rice straw was improved by the display of T. reeseii EGII using the novel gene cassette.ConclusionsWe have developed novel gene cassettes for the efficient cell-surface display of exo- and endo-type cellulolytic enzymes. The results suggest that this gene cassette has the wide applicability for cell-surface display and that cellulase-displaying yeasts have significant potential for cost-effective bioethanol production from lignocellulosic biomass.

Enhanced cell‐surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell‐surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α‐mating pheromone (MFα1SP) were constructed for cell‐surface display of Aspergillus aculeatus β‐glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell‐surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3‐ and 1.9‐fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell‐surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358–2366.

Complete Genome Sequence of Kluyveromyces marxianus NBRC1777, a Nonconventional Thermotolerant Yeast

ABSTRACT We determined the genome sequence of the thermotolerant yeast Kluyveromyces marxianus strain NBRC1777. The genome of strain NBRC1777 is composed of 4,912 open reading frames (ORFs) on 8 chromosomes, with a total size of 10,895,581 bp, including mitochondrial DNA.

Mechanical milling and membrane separation for increased ethanol production during simultaneous saccharification and co-fermentation of rice straw by xylose-fermenting Saccharomyces cerevisiae

Mechanical milling and membrane separation were applied to simultaneous saccharification and co-fermentation from hydrothermally pretreated rice straw. Mechanical milling with minimized 4 cycles enabled 37.5±3.4gL(-1) and 45.3±4.4gL(-1) of ethanol production after 48h by xylose-fermenting Saccharomyces cerevisiae from solid fractions (200 and 250gL(-1)) of pretreated rice straw with 5 filter paper unitg-biomass(-1) cellulase (respectively, 77.3±7.1% and 74.7±7.3% of theoretical ethanol yield). Use of a membrane-based process including nanofiltration and ultrafiltration increased the sugar concentrations in the liquid fraction of pretreated rice straw and addition of this liquid fraction to 250gL(-1) solid fraction increased ethanol production to 52.0±0.4gL(-1) (73.8±0.6% of theoretical ethanol yield). Mechanical milling was effective in increasing enzymatic hydrolysis of the solid fraction and membrane separation steps increased the ethanol titer during co-fermentation, leading to a proposal for combining these processes for ethanol production from whole rice straw.

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell‐surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201–1207.

Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production

Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a “tearing” cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions provided a novel strategy for increasing cellulose hydrolysis, suggesting a mechanism for promoting the feasibility of cellulosic biofuel production.

Mapping of endoglucanases displayed on yeast cell surface using atomic force microscopy

The surface of yeast cells has been an attractive interface for the effective use of cellulose. Surface enzymes, however, are difficult to visualize and evaluate. In this study, two kinds of unique anchoring regions were used to display the cellulase, endoglucanase (EG), on a yeast cell surface. Differences in the display level and the localization of EG were observed by atomic force microscopy. By surveying the yeast cell surface with a chemically modified cantilever, the interactive force between the cellulose and EG was measured. Force curve mapping revealed differences in the display levels and the localization of EG according to anchoring regions. The proposed methodology enables visualization of displayed enzymes such as EG on the yeast cell surface.

Widespread effect of N -acetyl- d -glucosamine assimilation on the metabolisms of amino acids, purines, and pyrimidines in Scheffersomyces stipitis

BackgroundFollowing cellulose, chitin is the most abundant renewable resource and is composed of the monomeric amino sugar N-acetyl-d-glucosamine (GlcNAc). Although many yeasts, including Saccharomyces cerevisiae, have lost their ability to utilize GlcNAc, some yeasts are able to use GlcNAc as a carbon source. However, our understanding of the effects of GlcNAc on the intracellular metabolism of nitrogen-containing compounds in these yeast species is limited.ResultsIn the present study, we quantitatively investigated the metabolic responses to GlcNAc in the GlcNAc-assimilating yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The comprehensive analysis of the metabolites extracted from S. stipitis cells grown in glucose, xylose, or GlcNAc revealed increased intracellular accumulation of a wide range of nitrogen-containing compounds during GlcNAc assimilation in this yeast. The levels of aromatic, branched-chain, and sulfur-containing amino acids and adenine, guanine, and cytosine nucleotides were the highest in GlcNAc-grown cells.ConclusionsThe CE-TOFMS analysis revealed a positive effect for GlcNAc on the intracellular concentration of a wide range of nitrogen-containing compounds. The metabolomic data gathered in this study will be useful for designing effective genetic engineering strategies to develop novel S. stipitis strains for the production of valuable nitrogen-containing compounds from GlcNAc.

Improvement of Xylose Fermentation Ability under Heat and Acid Co-Stress in Saccharomyces cerevisiae Using Genome Shuffling Technique

Xylose-assimilating yeasts with tolerance to both fermentation inhibitors (such as weak organic acids) and high temperature are required for cost-effective simultaneous saccharification and cofermentation (SSCF) of lignocellulosic materials. Here, we demonstrate the construction of a novel xylose-utilizing Saccharomyces cerevisiae strain with improved fermentation ability under heat and acid co-stress using the drug resistance marker-aided genome shuffling technique. The mutagenized genome pools derived from xylose-utilizing diploid yeasts with thermotolerance or acid tolerance were shuffled by sporulation and mating. The shuffled strains were then subjected to screening under co-stress conditions of heat and acids, and the hybrid strain Hyb-8 was isolated. The hybrid strain displayed enhanced xylose fermentation ability in comparison to both parental strains under co-stress conditions of heat and acids. Hyb-8 consumed 33.1 ± 0.6 g/L xylose and produced 11.1 ± 0.4 g/L ethanol after 72 h of fermentation at 38°C with 20 mM acetic acid and 15 mM formic acid. We also performed transcriptomic analysis of the hybrid strain and its parental strains to screen for key genes for multiple stress tolerances. We found that 13 genes, including 5 associated with cellular transition metal ion homeostasis, were significantly upregulated in Hyb-8 compared to levels in both parental strains under co-stress conditions. The hybrid strain Hyb-8 has strong potential for cost-effective SSCF of lignocellulosic materials. Moreover, the transcriptome data gathered in this study will be useful for understanding the mechanisms of multiple tolerance to high temperature and acids in yeast and facilitate the development of robust yeast strains for SSCF.