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Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.
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Glucósidos , Yarrowia , Glucósidos/metabolismo , Yarrowia/genética , Ingeniería Metabólica/métodos , FermentaciónRESUMEN
The market size of biosurfactants (BSs) has been expanding at an extremely fast pace due to their broad application scope. Therefore, the re-construction of cell factories with modified genomic and metabolic profiles for desired industrial performance has been an intriguing aspect. Typical mutagenesis approaches generate huge mutant libraries, whereas a battery of specific, robust, and cost-effective high-throughput screening (HTS) methods is requisite to screen target strains for desired phenotypes. So far, only a few specialized HTS assays have been developed for BSs that were successfully applied to obtain anticipated mutants. The most important milestones to reach, however, continue to be: specificity, sensitivity, throughput, and the potential for automation. Here, we discuss important colorimetric and fluorometric HTS approaches for possible intervention on automated HTS platforms. Moreover, we explain current bottlenecks in developing specialized HTS platforms for screening high-yielding producers and discuss possible perspectives for addressing such challenges.
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BACKGROUND: In the recombinant protein market with broad economic value, the rapid development of synthetic biology has made it necessary to construct an efficient exocrine expression system for the different heterologous proteins. Yarrowia lipolytica possesses unique advantages in nascent protein transport and glycosylation modification, so it can serve as a potential protein expression platform. Although the Po1 series derived from W29 is often used for the expression of the various heterologous proteins, the ability of W29 to secrete proteins has not been verified and the Po1 series has been found to be not convenient for further gene editing. RESULTS: A total of 246 Y. lipolytica strains were evaluated for their secretory capacity through performing high-throughput screening in 48-well plate. Thereafter, following two rounds of shake flask re-screening, a high-secreting protein starting strain DBVPG 5851 was obtained. Subsequently, combined with the extracellular protein types and relative abundance information provided by the secretome of the starting strain, available chassis cell for heterologous protein expression were preliminarily constructed, and it was observed that the most potential signal peptide was derived from YALI0D20680g. CONCLUSIONS: This study offers a novel perspective on the diversification of Y. lipolytica host cells for the heterologous protein expression and provides significant basis for expanding the selection space of signal peptide tools in the future research.
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Yarrowia , Yarrowia/genética , Secretoma , Ensayos Analíticos de Alto Rendimiento , Glicosilación , Proteínas Recombinantes/genéticaRESUMEN
Methyl ketones (MK) are highly valuable fatty acid derivatives with broad applications. Microbes based biosynthesis represents an alternative route for production of these usually fossil based chemicals. In this study, we reported metabolic engineering of Saccharomyces cerevisiae to produce MK, including 2-nonanone, 2-undecanone, 2-tridecanone and 2-pentadecanone. Besides enhancing inherent peroxisomal fatty acids ß-oxidation cycle, a novel heterologous cytosolic fatty acids ß-oxidation pathway was constructed, and this resulted in an increased production of MK by 2-fold. To increase carbon fluxes to methyl ketones, the supply of precursors was enhanced by engineering lipid metabolism, including improving the intracellular biosynthesis of acyl-CoAs, weakening the consumption of acyl-CoAs for lipids storage, and reinforcing activation of free fatty acids to acyl-CoAs. Hereby the titer of MK was improved by 7-fold, reaching 143.72 mg/L. Finally, transcription factor engineering was employed to increase the biosynthesis of methyl ketones and it was found that overexpression of ADR1 can mimic the oleate activated biogenesis and proliferation of peroxisomes, which resulted in a further increased production of MK by 28%. With these modifications and optimization, up to 845 mg/L total MK were produced from glucose in fed-batch fermentation, which is the highest titer of methyl ketones reported produced by fungi.
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Saccharomyces cerevisiae , Factores de Transcripción , Acetona/metabolismo , Ácidos Grasos/metabolismo , Cetonas/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable production of terpenoids. The non-conventional yeast, Yarrowia lipolytica, is a promising host for terpenoid biosynthesis due to its inherent mevalonate pathway, high fluxes of acetyl-CoA and NADPH, and the naturally hydrophobic microenvironment. In this review, we highlight progress in the engineering of Y. lipolytica as terpenoid biomanufacturing factories, describing the different terpenoid biosynthetic pathways and summarizing various metabolic engineering strategies, including progress in genetic manipulation, dynamic regulation, organelle engineering, and terpene synthase variants.
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Yarrowia , Acetilcoenzima A/metabolismo , Ingeniería Metabólica/métodos , Biología Sintética , Terpenos/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMEN
BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.
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Adenosina Trifosfato/metabolismo , Clostridium acetobutylicum/metabolismo , Fermentación , Glucólisis , Solventes/metabolismo , Acetona/metabolismo , Anaerobiosis , Biocombustibles , Butanoles/metabolismo , Clostridium acetobutylicum/genética , Etanol/metabolismo , HidrólisisRESUMEN
Pyruvate formate lyase (PFL) is characterized as an enzyme functional at anaerobic conditions, since the radical in the enzyme's active form is sensitive to oxygen. In this study, PFL and its activating enzyme from Escherichia coli were expressed in a Saccharomyces cerevisiae strain lacking pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc(-) MTH1-ΔT ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression of either of these electron donors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenases. Among the two electron donors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.
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Acetiltransferasas/biosíntesis , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Aerobiosis , Biomasa , Escherichia coli/enzimología , Escherichia coli/genética , Ferredoxinas/metabolismo , Flavodoxina/metabolismo , Perfilación de la Expresión Génica , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Yarrowia lipolytica has been widely used in the food biotech-related industry, where it plays the host's role in producing erythritol. Nevertheless, a temperature of about 28°C-30°C has been estimated as the yeast's optimal growth temperature, leading to the consumption of a considerable quantity of cooling water, especially in summer, which is obligatory for fermentation. Herein is described a method for improving the thermotolerance and erythritol production efficiency at high temperatures of Y. lipolytica. Through screening and testing different heat resistant devices, eight refactored engineered strains showed better growth at higher temperature and the antioxidant properties of the eight engineered strains were also improved. In addition, the erythritol titer, yield and productivity of the strain FOS11-Ctt1 represented the best among the eight strains, reaching at 39.25 g/L, 0.348 g/g glucose, and 0.55 g/L/h respectively, which were increased by 156%, 86% and 161% compared with the control strain, respectively. This study provides insight into an effective heat-resistant device that could enhance the thermotolerance and erythritol production of Y. lipolytica, which might be considered a valued scientific reference for other resistant strains' construction.
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Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO2 employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.
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Alcoholes del Azúcar , Azúcares , Alcoholes del Azúcar/metabolismo , Xilitol/metabolismo , Manitol/metabolismo , Eritritol/metabolismoRESUMEN
BACKGROUND: Owing to the Crabtree effect, Saccharomyces cerevisiae produces a large amount of ethanol in the presence of oxygen and excess glucose, leading to a loss of carbon for the biosynthesis of non-ethanol chemicals. In the present study, the potential of a newly constructed Crabtree negative S. cerevisiae, as a chassis cell, was explored for the biosynthesis of various non-ethanol compounds. RESULTS: To understand the metabolic characteristics of Crabtree negative S. cerevisiae sZJD-28, its transcriptional profile was compared with that of Crabtree positive S. cerevisiae CEN.PK113-11C. The reporter GO term analysis showed that, in sZJD-28, genes associated with translational processes were down-regulated, while those related to carbon metabolism were significantly up-regulated. To verify a potential increase in carbon metabolism for the Crabtree negative strain, the production of non-ethanol chemicals, derived from different metabolic nodes, was then undertaken for both sZJD-28 and CEN.PK113-11C. At the pyruvate node, production of 2,3-butanediol and lactate in sZJD-28-based strains was remarkably higher than that of CEN.PK113-11C-based ones, representing 16.8- and 1.65-fold increase in titer, as well as 4.5-fold and 0.65-fold increase in specific titer (mg/L/OD), respectively. Similarly, for shikimate derived p-coumaric acid, the titer of sZJD-28-based strain was 0.68-fold higher than for CEN.PK113-11C-based one, with a 0.98-fold increase in specific titer. While farnesene and lycopene, two acetoacetyl-CoA derivatives, showed 0.21- and 1.88-fold increases in titer, respectively. From malonyl-CoA, the titer of 3-hydroxypropionate and fatty acids in sZJD-28-based strains were 0.19- and 0.76-fold higher than that of CEN.PK113-11C-based ones, respectively. In fact, yields of products also improved by the same fold due to the absence of residual glucose. Fed-batch fermentation further showed that the titer of free fatty acids in sZJD-28-based strain 28-FFA-E reached 6295.6 mg/L with a highest reported specific titer of 247.7 mg/L/OD in S. cerevisiae. CONCLUSIONS: Compared with CEN.PK113-11C, the Crabtree negative sZJD-28 strain displayed a significantly different transcriptional profile and obvious advantages in the biosynthesis of non-ethanol chemicals due to redirected carbon and energy sources towards metabolite biosynthesis. The findings, therefore, suggest that a Crabtree negative S. cerevisiae strain could be a promising chassis cell for the biosynthesis of various chemicals.
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Dietary restriction of l-methionine, an essential amino acid, exerts potent antitumor effects on l-methionine-dependent cancers. However, dietary restriction of l-methionine has not been practical for human therapy because of the problem with the administration of l-methionine concentration in foods. Here, a thermophilic methionine γ-lyase (MGL), that catalyzes the cleavage of the C-S bond in l-methionine to produce α-ketobutyric acid, methanethiol, and ammonia was engineered from human cystathionine γ-lyase and almost completely depleted l-methionine at 65 °C, a temperature that accelerates the volatilization of methanethiol and its oxidation products. The high efficiency of l-methionine lysis may be attributed to the cooperative fluctuation and moderate the structural rigidity of 4 monomers in the thermophilic MGL, which facilitates l-methionine access to the entrance of the active site. Experimental diets treated with thermophilic MGL markedly inhibited prostate tumor growth in mice, and in parallel, the in vivo concentrations of l-methionine, its transformation product l-cysteine, and the oxidative stress indicator malondialdehyde significantly decreased. These findings provide a technology for the depletion of l-methionine in foods with an engineered thermophilic MGL, which efficiently inhibits tumor growth in mice.
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Sophorolipids are highly active green surfactants (glycolipid biosurfactants) getting tremendous appreciation worldwide due to their low toxicity, biodegradability, broad spectrum of applications, and significant biotechnological potential. Sophorolipids are mainly produced by an oleaginous budding yeast Starmerella bombicola using low-cost substrates. Therefore, the recent state-of-art literature information about S. bombicola yeast is hereby provided, especially the underlying production pathways, biosynthetic gene cluster, and regulatory enzymes. Moreover, the S. bombicola offers flexibility for regulating the structural diversity of sophorolipids, either genetically or by varying fermentative conditions. The emergence of advanced technologies like 'Omics and CRISPR/Cas have certainly boosted rational engineering research for designing high-performing platform strains. Therefore, currently available genetic engineering tools in S. bombicola were reviewed, thereby opening up exciting new possibilities for improving the overall bioproduction titers, structural variability, and stability of sophorolipids. Finally, some technical perspectives to address the current challenges were discussed.
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Saccharomycetales , Glucolípidos , Ácidos Oléicos , Saccharomycetales/genética , LevadurasRESUMEN
Saccharomyces cerevisiae, as a Generally Recognized as Safe (GRAS) fungus, has become one of the most widely used chassis cells for industrial applications and basic research. However, owing to its complex genetic background and intertwined metabolic networks, there are still many obstacles that need to be overcome in order to improve desired traits and to successfully link genotypes to phenotypes. In this context, genome editing and evolutionary technology have rapidly progressed over the last few decades to facilitate the rapid generation of tailor-made properties as well as for the precise determination of relevant gene targets that regulate physiological functions, including stress resistance, metabolic-pathway optimization and organismal adaptation. Directed genome evolution has emerged as a versatile tool to enable researchers to access desired traits and to study increasingly complicated phenomena. Here, the development of directed genome evolutions in S. cerevisiae is reviewed, with a focus on different techniques driving evolutionary engineering.
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Nuclease based genome editing systems have emerged as powerful tools to drive genomic alterations and enhance genome evolution via precise engineering in the various human and microbial cells. However, error-prone DNA repair has not been well studied previously to generate diverse genomic alterations and novel phenotypes. Here, we systematically investigated the potential interplay between DNA double strand break (DSB) repair and genome editing tools, and found that modulating the DSB end resection proteins could significantly improve mutational efficiency and diversity without exogenous DNA template in yeast. Deleting SAE2, EXO1, or FUN30, or overexpressing MRE11-H125N (nuclease-dead allele of MRE11), for DSB end resection markedly increased the efficiency of CRISPR/SpCas9 (more than 22-fold) and CRISPR/AsCpf1 (more than 30-fold)-induced mutagenesis. Deleting SAE2 or overexpressing MRE11-H125N substantially diversified CRISPR/SpCas9 or AsCpf1-induced mutation 2-3-fold at URA3 locus, and 3-5-fold at ADE2 locus. Thus, the error-prone DNA repair protein was employed to develop a novel mutagenic genome editing (mGE) strategy, which can increase the mutation numbers and effectively improve the ethanol/glycerol ratio of Saccharomyces cerevisiae through modulating the expression of FPS1 and GPD1. This study highlighted the feasibility of potentially reshaping the capability of genome editing by regulating the different DSB repair proteins and can thus expand the application of genome editing in diversifying gene expression and enhancing genome evolution. IMPORTANCE Most of the published papers about nuclease-assisted genome editing focused on precision engineering in human cells. However, the topic of inducing mutagenesis via error-prone repair has often been ignored in yeast. In this study, we reported that perturbing DNA repair, especially modifications of the various DSB end resection-related proteins, could greatly improve the mutational efficiency and diversity, and thus functionally reshape the capability of the different genome editing tools without requiring an exogenous DNA template in yeast. Specifically, mutagenic genome editing (mGE) was developed based on CRISPR/AsCpf1 and MRE11-H125N overexpression, and used to generate promoters of different strengths more efficiently. Thus, this work provides a novel method to diversify gene expression and enhance genome evolution.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Endonucleasas/genética , Endonucleasas/metabolismo , Genómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Yarrowia lipolytica is a widely-used chassis cell in biotechnological applications. It has recently gained extensive research interest owing to its extraordinary ability of producing industrially valuable biochemicals from a variety of carbon sources. Genome-scale metabolic models (GSMMs) enable analyses of cellular metabolism for engineering various industrial hosts. In the present study, we developed a high-quality GSMM iYli21 for Y. lipolytica type strain W29 by extensive manual curation with Biolog experimental data. The model showed a high accuracy of 85.7% in predicting nutrient utilization. Transcriptomics data were integrated to delineate cellular metabolism of utilizing six individual metabolites as sole carbon sources. Comparisons showed that 302 reactions were commonly used, including those from TCA cycle, oxidative phosphorylation, and purine metabolism for energy and material supply. Whereas glycolytic reactions were employed only when glucose and glycerol used as sole carbon sources, gluconeogenesis and fatty acid oxidation reactions were specifically employed when fatty acid, alkane and glycerolipid were the sole carbon sources. Further test of 46 substrates for generating 5 products showed that hexanoate outcompeted other compounds in terms of maximum theoretical yield owing to the lowest carbon loss for energy supply. This newly generated model iYli21 will be a valuable tool in dissecting metabolic mechanism and guiding metabolic engineering of this important industrial cell factory.
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Non-conventional yeasts have attracted a growing interest on account of their excellent characteristics. In recent years, the emerging of CRISPR/Cas technology has improved the efficiency and accuracy of genome editing. Utilizing the advantages of CRISPR/Cas in bioengineering of non-conventional yeasts, quite a few advancements have been made. Due to the diversity in their genetic background, the ways for building a functional CRISPR/Cas system of various species non-conventional yeasts were also species-specific. Herein, we have summarized the different strategies for optimizing CRISPR/Cas systems in different non-conventional yeasts and their biotechnological applications in the construction of cell factories. In addition, we have proposed some potential directions for broadening and improving the application of CRISPR/Cas technology in non-conventional yeasts.
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Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.
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Ingeniería Metabólica , Yarrowia , Edición Génica , Pichia/genética , Biología Sintética , Yarrowia/genética , LevadurasRESUMEN
Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.
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Regulación Fúngica de la Expresión Génica , Redes y Vías Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Perfilación de la Expresión Génica , Glucosa/metabolismo , Ingeniería Metabólica , Mutación , Piruvato Descarboxilasa/genética , Ácido Pirúvico/metabolismo , ARN Polimerasa II/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Biología de SistemasRESUMEN
Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.
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Alcohol Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa/metabolismo , Reactores Biológicos/microbiología , Butanoles/metabolismo , Clostridium acetobutylicum , Etanol/metabolismo , Fermentación/genética , Alcohol Deshidrogenasa/genética , Aldehído Deshidrogenasa/genética , Biocombustibles/microbiología , Clostridium acetobutylicum/enzimología , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , ADN Bacteriano/genética , Fermentación/fisiología , Oxidación-ReducciónRESUMEN
Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.