Engineering of Saccharomyces cerevisiae for efficient fermentation of cellulose.

Conversion of lignocellulosic biomass to biofuels using microbial fermentation is an attractive option to substitute petroleum-based production economically and sustainably. The substantial efforts to design yeast strains for biomass hydrolysis have led to industrially applicable biological routes. Saccharomyces cerevisiae is a robust microbial platform widely used in biofuel production, based on its amenability to systems and synthetic biology tools. The critical challenges for the efficient microbial conversion of lignocellulosic biomass by engineered S. cerevisiae include heterologous expression of cellulolytic enzymes, co-fermentation of hexose and pentose sugars, and robustness against various stresses. Scientists developed many engineering strategies for cellulolytic S. cerevisiae strains, bringing the application of consolidated bioprocess at an industrial scale. Recent advances in the development and implementation of engineered yeast strains capable of assimilating lignocellulose will be reviewed.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

Direct conversion of cellulose into ethanol and ethyl-ß-d-glucoside via engineered Saccharomyces cerevisiae.

Simultaneous saccharification and fermentation (SSF) of cellulose via engineered Saccharomyces cerevisiae is a sustainable solution to valorize cellulose into fuels and chemicals. In this study, we demonstrate the feasibility of direct conversion of cellulose into ethanol and a biodegradable surfactant, ethyl-ß-d-glucoside, via an engineered yeast strain (i.e., strain EJ2) expressing heterologous cellodextrin transporter (CDT-1) and intracellular ß-glucosidase (GH1-1) originating from Neurospora crassa. We identified the formation of ethyl-ß-d-glucoside in SSF of cellulose by the EJ2 strain owing to transglycosylation activity of GH1-1. The EJ2 strain coproduced 0.34 ± 0.03 g ethanol/g cellulose and 0.06 ± 0.00 g ethyl-ß-d-glucoside/g cellulose at a rate of 0.30 ± 0.02 g·L-1 ·h-1 and 0.09 ± 01 g·L-1 ·h-1 , respectively, during the SSF of Avicel PH-101 cellulose, supplemented only with Celluclast 1.5 L. Herein, we report a possible coproduction of a value-added chemical (alkyl-glucosides) during SSF of cellulose exploiting the transglycosylation activity of GH1-1 in engineered S. cerevisiae. This coproduction could have a substantial effect on the overall technoeconomic feasibility of theSSF of cellulose.

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

UNLABELLED: Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCE: In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.

2,3-butanediol production from cellobiose by engineered Saccharomyces cerevisiae.

Production of renewable fuels and chemicals from cellulosic biomass is a critical step towards energy sustainability and reduced greenhouse gas emissions. Microbial cells have been engineered for producing chemicals from cellulosic sugars. Among these chemicals, 2,3-butanediol (2,3-BDO) is a compound of interest due to its diverse applications. While microbial production of 2,3-BDO with high yields and productivities has been reported, there are concerns associated with utilization of potential pathogenic bacteria and inefficient utilization of cellulosic sugars. To address these problems, we engineered 2,3-BDO production in Saccharomyces cerevisiae, especially from cellobiose, a prevalent sugar in cellulosic hydrolysates. Specifically, we overexpressed alsS and alsD from Bacillus subtilis to convert pyruvate into 2,3-BDO via α-acetolactate and acetoin in an engineered cellobiose fermenting S. cerevisiae. Under oxygen-limited conditions, the resulting strain was able to produce 2,3-BDO. Still, major carbon flux went to ethanol, resulting in substantial amounts of ethanol produced as a byproduct. To enhance pyruvate flux to 2,3-BDO through elimination of the pyruvate decarboxylation reaction, we employed a deletion mutant of both PDC1 and PDC5 for producing 2,3-BDO. When a cellobiose utilization pathway, consisting of a cellobiose transporter and intracellular ß-glucosidase, and the 2,3-BDO producing pathway were introduced in a pyruvate decarboxylase deletion mutant, the resulting strain produced 2,3-BDO without ethanol production from cellobiose under oxygen-limited conditions. A titer of 5.29 g/l 2,3-BDO with a productivity of 0.22 g/l h and yield of 0.29 g 2,3-BDO/g cellobiose was attained. These results suggest the possibility of producing 2,3-BDO safely and sustainably from cellulosic hydrolysates.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae.

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae.

As Saccharomyces cerevisiae cannot utilize xylose as a carbon source, expression of XYL1 coding for xylose reductase (XR) from Scheffersomyces (Pichia) stipitis enabled production of xylitol from xylose with a high yield. However, insufficient supply of NAD(P)H for XR and inhibition of xylose uptake by glucose are identified as major constraints for achieving high xylitol productivity. To overcome these problems, we engineered S. cerevisiae capable of converting xylose into xylitol through simultaneous utilization of xylose and cellobiose. An engineered S. cerevisiae (D-10-BT) expressing XR, cellodextrin transporter (cdt-1) and intracellular ß-glucosidase (gh1-1) produced xylitol via simultaneous utilization of cellobiose and xylose. The D-10-BT strain exhibited 40% higher volumetric xylitol productivity with co-consumption of cellobiose and xylose compared to sequential utilization of glucose and xylose. Furthermore, the overexpression of S. cerevisiae ALD6, IDP2, or S. stipitis ZWF1 coding for cytosolic NADP(+)-dependent dehydrogenases increased the intracellular NADPH availability of the D-10-BT strain, which resulted in a 37-63% improvement in xylitol productivity when cellobiose and xylose were co-consumed. These results suggest that co-utilization of cellobiose and xylose can lead to improved xylitol production through enhanced xylose uptake and efficient cofactor regeneration.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Recent advances in the biological valorization of citrus peel waste into fuels and chemicals.

In the quest to reduce global food loss and waste, fruit processing wastes, particularly citrus peel waste (CPW), have emerged as a promising and sustainable option for biorefinery without competing with human foods and animal feeds. CPW is largely produced and, as recent studies suggest, has the industrial potential of biological valorization into fuels and chemicals. In this review, the promising aspects of CPW as an alternative biomass were highlighted, focusing on its low lignin content. In addition, specific technical difficulties in fermenting CPW are described, highlighting that citrus peel is high in pectin that consist of non-fermentable sugars, mainly galacturonic acid. Last, recent advances in the metabolic engineering of yeast and other microbial strains that ferment CPW-derived sugars to produce value-added products, such as ethanol and mucic acid, are summarized. For industrially viable CPW-based biorefinery, more studies are needed to improve fermentation efficiency and to diversify product profiles.

The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels.

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

Redirection of the Glycolytic Flux Enhances Isoprenoid Production in Saccharomyces cerevisiae.

Sufficient supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a prerequisite of the overproduction of isoprenoids and related bioproducts in Saccharomyces cerevisiae. Although S. cerevisiae highly depends on the oxidative pentose phosphate (PP) pathway to produce NADPH, its metabolic flux toward the oxidative PP pathway is limited due to the rigid glycolysis flux. To maximize NADPH supply for the isoprenoid production in yeast, upper glycolytic metabolic fluxes are reduced by introducing mutations into phosphofructokinase (PFK) along with overexpression of ZWF1 encoding glucose-6-phosphate (G6P) dehydrogenase. The PFK mutations (Pfk1 S724D and Pfk2 S718D) result in less glycerol production and more accumulation of G6P, which is a gateway metabolite toward the oxidative PP pathway. When combined with the PFK mutations, overexpression of ZWF1 caused substantial increases of [NADPH]/[NADP+ ] ratios whereas the effect of ZWF1 overexpression alone in the wild-type strain is not noticeable. Also, the introduction of ZWF1 overexpression and the PFK mutations into engineered yeast overexpressing acetyl-CoA C-acetyltransferase (ERG10), truncated HMG-CoA reductase isozyme 1 (tHMG1), and amorphadiene synthase (ADS) leads to a titer of 497 mg L-1 of amorphadiene (3.7-fold over the parental strain). These results suggest that perturbation of upper glycolytic fluxes, in addition to ZWF1 overexpression, is necessary for efficient NADPH supply through the oxidative PP pathway and enhanced production of isoprenoids by engineered S. cerevisiae.

Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform.

Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae.

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.

Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.

In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

Overexpression of RCK1 improves acetic acid tolerance in Saccharomyces cerevisiae.

Mixed sugars derived from lignocellulosic biomass can be converted into biofuels and chemicals by engineered microorganisms, but toxic fermentation inhibitors produced from harsh depolymerization processes of lignocellulosic biomass pose a critical challenge for economic production of biofuels and chemicals. Unlike other fermentation inhibitors generated from sugar degradation, acetic acid is inevitably produced from acetylated hemicellulose, and its concentrations in cellulosic hydrolysates are substantially higher than other fermentation inhibitors. The aim of this study was to identify novel genetic perturbations for improved acetic acid tolerance in Saccharomyces cerevisiae. Through a genomic library-based approach, we identified an overexpression gene target RCK1 coding for a protein kinase involved in oxidative stress. Overexpression of RCK1 significantly improved glucose and xylose fermentation under acetic acid stress conditions. Specifically, the RCK1-overexpressing strain exhibited a two-fold higher specific ethanol productivity than the control strain in glucose fermentation under the presence of acetic acid. Interestingly, the engineered S. cerevisiae overexpressing RCK1 showed 40% lower intracellular reactive oxygen species (ROS) levels as compared to the parental strain when the strains were exposed to acetic acid, suggesting that RCK1 overexpression might play a role in reducing the oxidative stress caused by acetic acid.