scholarly journals Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

2016 ◽  
Vol 82 (7) ◽  
pp. 2156-2166 ◽  
Author(s):  
Yingying Chen ◽  
Lisa Stabryla ◽  
Na Wei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Metabolic Engineering ◽  
Acid Stress ◽  
Acid Resistance ◽  
Biofuel Production ◽  
Gene Target ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

scholarly journals Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

2014 ◽  
Vol 80 (17) ◽  
pp. 5542-5550 ◽  
Author(s):  
Zihe Liu ◽  
Lifang Liu ◽  
Tobias Österlund ◽  
Jin Hou ◽  
Mingtao Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Secretory Pathway ◽  
Single Point ◽  
Point Mutations ◽  
Model Organisms ◽  
Cell Factory ◽  
Amylase Secretion ◽  
Content Type ◽  
Inverse Metabolic Engineering

ABSTRACTThe increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeastSaccharomyces cerevisiaeis widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in theVTA1gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

scholarly journals Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

2005 ◽  
Vol 71 (12) ◽  
pp. 8249-8256 ◽  
Author(s):  
Yong-Su Jin ◽  
Hal Alper ◽  
Yea-Tyng Yang ◽  
Gregory Stephanopoulos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Metabolic Engineering ◽  
Ethanol Production ◽  
Pichia Stipitis ◽  
Pentose Phosphate ◽  
Gene Target ◽  
Reading Frame ◽  
Engineering Approach ◽  
Inverse Metabolic Engineering

ABSTRACT We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.

scholarly journals Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

2014 ◽  
Vol 80 (11) ◽  
pp. 3488-3495 ◽  
Author(s):  
Minetaka Sugiyama ◽  
Shin-Pei Akase ◽  
Ryota Nakanishi ◽  
Hitoshi Horie ◽  
Yoshinobu Kaneko ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Subcellular Localization ◽  
Target Genes ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Nuclear Accumulation ◽  
Content Type ◽  
Adaptation Response

ABSTRACTImprovement of the lactic acid resistance of the yeastSaccharomyces cerevisiaeis important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened forS. cerevisiaegenes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genesYGP1,GPG1, andSPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin geneMSN5led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

scholarly journals Enhancement of Acetic Acid Tolerance in Saccharomyces cerevisiae by Overexpression of theHAA1Gene, Encoding a Transcriptional Activator

2012 ◽  
Vol 78 (22) ◽  
pp. 8161-8163 ◽  
Author(s):  
Koichi Tanaka ◽  
Yukari Ishii ◽  
Jun Ogawa ◽  
Jun Shima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Acetic Acid Tolerance ◽  
Content Type ◽  
In The Wild ◽  
Overexpressing Strain

ABSTRACTHaa1 is a transcriptional activator required forSaccharomyces cerevisiaeadaptation to weak acids. Here we show that the constitutiveHAA1-overexpressing strain acquired a higher level of acetic acid tolerance. Under conditions of acetic acid stress, the intracellular level of acetic acid was significantly lower inHAA1-overexpressing cells than in the wild-type cells.

scholarly journals The dual role of candida glabrata drug:H+ antiporter CgAqr1 (ORF CAGL0J09944g) in antifungal drug and acetic acid resistance

2013 ◽  
Vol 4 ◽  
Author(s):  
Catarina Costa ◽  
André Henriques ◽  
Carla Pires ◽  
Joana Nunes ◽  
Michiyo Ohno ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Candida Glabrata ◽  
Acid Resistance ◽  
Dual Role ◽  
Antifungal Drug ◽  
Acetic Acid Resistance

scholarly journals Endogenous lycopene improves ethanol production under acetic acid stress in Saccharomyces cerevisiae

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Pan ◽  
Bin Jia ◽  
Hong Liu ◽  
Zhen Wang ◽  
Meng-Zhe Chai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Ethanol Production ◽  
Acid Stress

scholarly journals Molecular Design of a Signaling System Influences Noise in Protein Abundance under Acid Stress in Different Gammaproteobacteria

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sophie Brameyer ◽  
Elisabeth Hoyer ◽  
Sebastian Bibinger ◽  
Korinna Burdack ◽  
Jürgen Lassak ◽  
...  
Keyword(s):  
Copy Number ◽  
Molecular Design ◽  
Ph Sensor ◽  
Acid Stress ◽  
Acid Resistance ◽  
Signaling System ◽  
Content Type ◽  
Signaling Systems ◽  
E Coli ◽  
Cad System

ABSTRACT Bacteria have evolved different signaling systems to sense and adapt to acid stress. One of these systems, the CadABC system, responds to a combination of low pH and lysine availability. In Escherichia coli, the two signals are sensed by the pH sensor and transcription activator CadC and the cosensor LysP, a lysine-specific transporter. Activated CadC promotes the transcription of the cadBA operon, which codes for the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. The copy number of CadC is controlled translationally. Using a bioinformatics approach, we identified the presence of CadC with ribosomal stalling motifs together with LysP in species of the Enterobacteriaceae family. In contrast, we identified CadC without stalling motifs in species of the Vibrionaceae family, and the LysP cosensor is missing. Therefore, we compared the outputs of the Cad system in single cells of the distantly related organisms E. coli and Vibrio campbellii using fluorescently tagged CadB as the reporter. We observed a heterogeneous output in E. coli, and all the V. campbellii cells produced CadB. The copy number of the pH sensor CadC in E. coli was extremely low (≤4 molecules per cell), but it was 10-fold higher in V. campbellii. An increase in the CadC copy number in E. coli correlated with a decrease in heterogeneous behavior. This study demonstrated how small changes in the design of a signaling system allow a homogeneous output and, thus, adaptation of Vibrio species that rely on the CadABC system as the only acid resistance system. IMPORTANCE Acid resistance is an important property for bacteria, such as Escherichia coli, to survive acidic environments like the human gastrointestinal tract. E. coli possesses both passive and inducible acid resistance systems to counteract acidic environments. Thus, E. coli evolved sophisticated signaling systems to sense and appropriately respond to environmental acidic stress by regulating the activity of its three inducible acid resistance systems. One of these systems is the Cad system, which is induced only under moderate acidic stress in a lysine-rich environment by the pH-responsive transcriptional regulator CadC. The significance of our research lies in identifying the molecular design of the Cad systems in different proteobacteria and their target expression noise at the single-cell level during acid stress conditions.

scholarly journals Putative ABC Transporter Responsible for Acetic Acid Resistance in Acetobacter aceti

2006 ◽  
Vol 72 (1) ◽  
pp. 497-505 ◽  
Author(s):  
Shigeru Nakano ◽  
Masahiro Fukaya ◽  
Sueharu Horinouchi
Keyword(s):  
Acetic Acid ◽  
Abc Transporter ◽  
Acid Resistance ◽  
Acetic Acid Bacteria ◽  
Electrophoretic Analysis ◽  
Nucleotide Sequencing ◽  
Multicopy Plasmid ◽  
Coding Region ◽  
Acetobacter Aceti ◽  
Acetic Acid Resistance

ABSTRACT Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH2-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.

Inverse metabolic engineering for improved galactose fermentation by Saccharomyces cerevisiae

2009 ◽  
Vol 108 ◽  
pp. S172-S173
Author(s):  
Ki-Sung Lee ◽  
Young-Je Sung ◽  
Yoon-Ha Hwang ◽  
Yong-Cheol Park ◽  
Jin-Ho Seo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Inverse Metabolic Engineering
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