Overcoming the Limitations of CRISPR-Cas9 Systems in Saccharomyces cerevisiae: Off-Target Effects, Epigenome, and Mitochondrial Editing.

Modification of the genome of the yeast Saccharomyces cerevisiae has great potential for application in biological research and biotechnological advancements, and the CRISPR-Cas9 system has been increasingly employed for these purposes. The CRISPR-Cas9 system enables the precise and simultaneous modification of any genomic region of the yeast to a desired sequence by altering only a 20-nucleotide sequence within the guide RNA expression constructs. However, the conventional CRISPR-Cas9 system has several limitations. In this review, we describe the methods that were developed to overcome these limitations using yeast cells. We focus on three types of developments: reducing the frequency of unintended editing to both non-target and target sequences in the genome, inducing desired changes in the epigenetic state of the target region, and challenging the expansion of the CRISPR-Cas9 system to edit genomes within intracellular organelles such as mitochondria. These developments using yeast cells to overcome the limitations of the CRISPR-Cas9 system are a key factor driving the advancement of the field of genome editing.

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases.

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing.

Development of Artificial System to Induce Chromatin Loosening in Saccharomyces cerevisiae.

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus.

Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Engineering, Bio-Arming Technology.

The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of protein/peptide library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-activated cell sorting (FACS), and yeast cell chip, the cell surface display system is an effective molecular tool for high-throughput screening of mutated protein/peptide library. In this article, we describe the methods for cell surface display of proteins/peptides of interest on yeast, evaluation of display efficiency, and harvesting of the displayed proteins/peptides from cell surface.

Simultaneous Display of Multiple Kinds of Enzymes on the Yeast Cell Surface for Multistep Reactions.

The yeast surface display system is a valuable platform for constructing cells with novel functions for various applications and high-throughput screening of protein or peptide libraries containing random mutations. Among the host microorganisms used for surface display, yeast is the most suitable microorganism for surface engineering owing to its eukaryotic features. In yeast, proper folding and glycosylation of expressed eukaryotic proteins can be performed. Furthermore, in this system, multiple kinds of proteins can be simultaneously displayed on the cell surface. This allows for a synergistic effect between the displayed enzymes, leading to an efficient multistep reaction. Alternatively, the ratio of the enzymes to be displayed can be controlled by the co-culture of surface-engineered yeasts displaying a single kind of enzyme. Therefore, yeast surface display systems have been applied to the construction of various whole-cell biocatalysts. Here, we describe methods for the simultaneous display of multiple kinds of proteins on the yeast cell surface.

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli.

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii.

Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae.

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae.

Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a "mito-CRISPR system" that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

CRISPR Nickase-Mediated Base Editing in Yeast.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5 days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.

Efficient ammonia production from food by-products by engineered Escherichia coli.

Ammonia is used as a fertilizer for agriculture, chemical raw material, and carrier for transporting hydrogen, and with economic development, the demand for ammonia has increased. The Haber-Bosch process, which is the main method for producing ammonia, can produce ammonia with high efficiency. However, since it consumes a large amount of fossil energy, it is necessary to develop an alternative method for producing ammonia with less environmental impact. Ammonia production from food by-products is an appealing production process owing to unused resource usage, including waste, and mild reaction conditions. However, when food by-products and biomass are used as feedstocks, impurities often reduce productivity. Using metabolic profiling, glucose was identified as a potential inhibitor of ammonia production from impure food by-products. We constructed the recombinant Escherichia coli, in which glucose uptake was reduced by ptsG gene disruption and amino acid catabolism was promoted by glnA gene disruption. Ammonia production efficiency from okara, a food by-product, was improved in this strain; 35.4 mM ammonia was produced (47% yield). This study might provide a strategy for efficient ammonia production from food by-products.

Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara).

Ammonia is an essential substance for agriculture and the chemical industry. The intracellular production of ammonia in yeast (Saccharomyces cerevisiae) by metabolic engineering is difficult because yeast strongly assimilates ammonia, and the knockout of genes enabling this assimilation is lethal. Therefore, we attempted to produce ammonia outside the yeast cells by displaying a glutaminase (YbaS) from Escherichia coli on the yeast cell surface. YbaS-displaying yeast successfully produced 3.34 g/L ammonia from 32.6 g/L glutamine (83.2% conversion rate), providing it at a higher yield than in previous studies. Next, using YbaS-displaying yeast, we also succeeded in producing ammonia from glutamine in soybean residues (okara) produced as food waste from tofu production. Therefore, ammonia production outside cells by displaying ammonia-lyase on the cell surface is a promising strategy for producing ammonia from food waste as a novel energy resource, thereby preventing food loss.

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides.

BACKGROUND: Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. RESULTS: We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. CONCLUSION: In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Direct bioethanol production from brown macroalgae by co-culture of two engineered Saccharomyces cerevisiae strains.

A co-culture platform for bioethanol production from brown macroalgae was developed, consisting of two types of engineered Saccharomyces cerevisiae strains; alginate- and mannitol-assimilating yeast (AM1), and cellulase-displaying yeast (CDY). When the 5% (w/v) brown macroalgae Ecklonia kurome was used as the sole carbon source for this system, 2.1 g/L of ethanol was produced, along with simultaneous consumption of alginate, mannitol, and glucans.

Xylanase B from Clostridium cellulovorans 743B: overexpression, purification, crystallization and X-ray diffraction analysis.

Clostridium cellulovorans produces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Šresolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 Å, α = ß = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans.

Platform construction of molecular breeding for utilization of brown macroalgae.

Brown macroalgae are characterized by a large size and high productivity without requiring arable land, fresh water, and fertilizer. Furthermore, since brown macroalgae contain little or no lignin, simple biorefinery processing can efficiently produce sugars from this material. Therefore, brown macroalgae have attracted attention as an alternative feedstock for bioethanol production. However, the utilization of biotechnologies previously developed for terrestrial biomass processing results in difficulties in the bioconversion of brown macroalgae. Recently, several studies have developed biotechnologies for using major carbohydrates of brown macroalgae, such as laminarin, mannitol, and alginate. This review focuses on these fermentation biotechnologies using natural or engineered microorganisms.

Erratum: Precise genome-wide base editing by the CRISPR Nickase system in yeast.

A correction to this Article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol.

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-L-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.