Single-Cell Phenotypic Screening in 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.

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Enabling inverse metabolic engineering through genomics

Current Opinion in Biotechnology ◽

10.1016/s0958-1669(03)00116-2 ◽

2003 ◽

Vol 14 (5) ◽

pp. 484-490 ◽

Cited By ~ 24

Author(s):

Ryan T Gill

Keyword(s):

Metabolic Engineering ◽

Inverse Metabolic Engineering

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

Applied and Environmental Microbiology ◽

10.1128/aem.03718-15 ◽

2016 ◽

Vol 82 (7) ◽

pp. 2156-2166 ◽

Cited By ~ 43

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.

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Recovery of Phenotypes Obtained by Adaptive Evolution through Inverse Metabolic Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01444-12 ◽

2012 ◽

Vol 78 (21) ◽

pp. 7579-7586 ◽

Cited By ~ 14

Author(s):

Kuk-Ki Hong ◽

Jens Nielsen

Keyword(s):

Metabolic Engineering ◽

Uptake Rate ◽

Complete Recovery ◽

System Level ◽

Inverse Metabolic Engineering ◽

Transcriptional Changes ◽

Galactose Utilization ◽

Yeast Mutants ◽

Reference Strains ◽

Level Analysis

ABSTRACTIn a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutationsRAS2Lys77,RAS2Tyr112, andERG5Pro370were constructed and evaluated. The mutants were also combined with overexpression ofPGM2, earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. TheRAS2Lys77mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. TheRAS2Tyr112mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. TheERG5Pro370mutation only showed a small improvement, but when it was combined withPGM2overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of theRAS2mutations withPGM2overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast.

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8249-8256.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8249-8256 ◽

Cited By ~ 110

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.

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