scholarly journals Improving isobutanol productivity through adaptive laboratory evolution in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Aili Zhang ◽  
Yide Su ◽  
Jingzhi Li ◽  
Weiwei Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Metabolic Engineering ◽  
Glucose Concentration ◽  
Evolutionary Engineering ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Whole Genome Resequencing

Abstract Background: Isobutanol is an ideal second-generation biofuels due to its lower hygroscopicity, higher energy density and higher-octane value. However, isobutanol is toxic to production organisms. To improve isobutanol productivity, adaptive laboratory evolution method was carried out to improve the tolerance of Saccharomyces cerevisiae toward higher isobutanol and higher glucose concentration.Results: We evolved the laboratory strain of S. cerevisiae W303-1A by using EMS (ethyl methanesulfonate) mutagenesis followed by adaptive laboratory evolution. The evolved strain EMS39 with significant increase in growth rate and viability in media with higher isobutanol and higher glucose concentration was obtained. Then, metabolic engineering of the evolved strain EMS39 as a platform for isobutanol production were carried out. Delta integration method was used to over-express ILV3 gene and 2μ plasmids carrying ILV2, ILV5 and ARO10 were used to over-express ILV2, ILV5 and ARO10 genes in the evolved strain EMS39 and wild type W303-1A. And the resulting strains was designated as strain EMS39V2δV3V5A10 and strain W303-1AV2δV3V5A10, respectively. Our results shown that isobutanol titers of the evolved strain EMS39 increased by 30% compared to the control strain. And isobutanol productivity of strain EMS39V2δV3V5A10 increased by 32.4% compared to strain W303-1AV2δV3V5A10. Whole genome resequencing and analysis of site-directed mutagenesis of the evolved strain EMS39 have identified important mutations. In addition, RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from EMS39.Conclusions: With the aim of increase productivity of isobutanol in S. cerevisiae, improving tolerance toward higher isobutanol and higher glucose concentration via EMS mutagenesis followed by adaptive evolutionary engineering was conducted. An evolved strain EMS39 with significant increase in growth rate and viability had been obtained. And metabolic engineering of the evolved strain as a platform for isobutanol production was carried out. Furthermore, analysis of whole genome resequencing and transcriptome sequencing were also carried out.

scholarly journals Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Buli Su ◽  
Anzhang Li ◽  
Ming-Rong Deng ◽  
Honghui Zhu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Selective Pressure ◽  
Large Family ◽  
Fold Increase ◽  
Loss Of Function ◽  
Genome Resequencing ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Whole Genome Resequencing ◽  
High Level

AbstractCarotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain.

scholarly journals Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Thomas Perli ◽  
Dewi P.I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
B Vitamin

AbstractQuantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

scholarly journals Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Thomas Perli ◽  
Dewi P. I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
B Vitamin

ABSTRACT Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.

scholarly journals Repetitive sequence variation and dynamics in the ribosomal DNA array of Saccharomyces cerevisiae as revealed by whole-genome resequencing

2009 ◽  
Vol 19 (4) ◽  
pp. 626-635 ◽  
Author(s):  
S. A. James ◽  
M. J.T. O'Kelly ◽  
D. M. Carter ◽  
R. P. Davey ◽  
A. van Oudenaarden ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Repetitive Sequence ◽  
Sequence Variation ◽  
Whole Genome ◽  
Dna Array ◽  
Genome Resequencing ◽  
Whole Genome Resequencing

Genetic diversity and population structure of Tibetan sheep breeds determined by whole genome resequencing

2021 ◽  
Vol 53 (1) ◽  
Author(s):  
Lei-Lei Li ◽  
Shi-Ke Ma ◽  
Wei Peng ◽  
You-Gui Fang ◽  
Hai-Rui Duo ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Sheep Breeds ◽  
Tibetan Sheep ◽  
Whole Genome Resequencing

scholarly journals Whole genome resequencing and custom genotyping unveil clonal lineages in ‘Malbec’ grapevines (Vitis vinifera L.)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Luciano Calderón ◽  
Nuria Mauri ◽  
Claudio Muñoz ◽  
Pablo Carbonell-Bejerano ◽  
Laura Bree ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Somatic Mutations ◽  
Clonal Propagation ◽  
Variant Calling ◽  
Vitis Vinifera L ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Genome Resequencing ◽  
Diversity Pattern ◽  
Whole Genome Resequencing

AbstractGrapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

Sign in / Sign up

Export Citation Format

Share Document