Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

Oud, Bart; Maris, Antonius J. A. van; Daran, Jean‐Marc; Pronk, Jack T.

doi:10.1111/j.1567-1364.2011.00776.x

Cited by 78 publications

(69 citation statements)

References 115 publications

(167 reference statements)

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“…To facilitate identification of mutations contributing to the multicellular phenotype (18,33), two identical independent anaerobic evolution experiments were started on a mixture of 20 g·L −1 glucose and 20 g·L −1 galactose. Although the specific growth rate on galactose doubled during both evolution experiments (from 0.11 to 0.22 and 0.20 h −1 ; Fig.…”

Section: Selection Of Multicellular Clusters In Sequential Bioreactormentioning

confidence: 99%

“…Laboratory evolution has generated new insights into mutation rates (11,12), genetic drift (12,13), epistasis (14), clonal interference (15), and other important aspects of evolution by natural selection (16). In microbial biotechnology, reverse engineering of evolved phenotypes, known as inverse metabolic engineering (17), has similarly benefited from the availability of these genomic methodologies (18). In this applied research context, knowledge of the genetic basis of an industrially relevant phenotype not only increases understanding, but also enables its reconstruction and improvement in other microbial strains and species (18)(19)(20).…”

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confidence: 99%

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Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Oud

Guadalupe-Medina

Nijkamp

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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101

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Significance The shift from unicellular to multicellular life forms represents a key innovation step in the evolution of life on Earth. However, knowledge on the evolutionary pressures resulting in the selection of multicellular life forms and the underlying molecular mechanisms is far from complete. Our study provides a complete identification of the specific genetic changes by which the unicellular eukaryote S. cerevisiae can acquire a multicellular, fast-sedimenting phenotype. We demonstrated that a minimal evolutionary mechanism encompassed a deregulation of the late step of the cell cycle through mutation in ACE2 followed by whole genome duplication.

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Section: Selection Of Multicellular Clusters In Sequential Bioreactormentioning

confidence: 99%

mentioning

confidence: 99%

Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Oud

Guadalupe-Medina

Nijkamp

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

101

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show abstract

“…Laboratory evolution under a particular selection condition and subsequent genome sequence analysis of a selected colony with the desired phenotype constitute a proven approach of inverse metabolic engineering (26,28). After comparing the genome sequences of strains EJ2 and EJ1, we identified only one nonsynonymous change in strain EJ2 compared to strain EJ1.…”

Section: Construction Of Cellobiose-utilizing Strains Via Integration Ofmentioning

confidence: 99%

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Skerker

Kim

et al. 2016

Appl Environ Microbiol

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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 ␤-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. IMPORTANCEIn 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 ␤-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 evol...

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“…Using the nanopore assembly as a reference for short-read sequencing of strains derived from CEN.PK113-7D will yield more complete and more accurate lists of SNPs and other mutations, facilitating the identification of causal mutations in laboratory evolution or mutagenesis experiments. Therefore, the new assembly should accelerate elucidation of the genetic basis underlying the fitness of S. cerevisiae in various environmental conditions, as well as the discovery of new strain improvement strategies for industrial applications (Oud et al 2012).…”

Section: Discussionmentioning

confidence: 99%

Nanopore sequencing enables near-completede novoassembly ofSaccharomyces cerevisiaereference strain CEN.PK113-7D

Salazar

Vries

Broek

et al. 2017

Preprint

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The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113-7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113-7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113-7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113-7D and places a caveat on assumed genome stability of microorganisms.

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Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

Cited by 78 publications

References 115 publications

Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Nanopore sequencing enables near-completede novoassembly ofSaccharomyces cerevisiaereference strain CEN.PK113-7D

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