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

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.

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

10.1101/2020.02.12.945287 ◽

2020 ◽

Cited By ~ 1

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.

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Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

Applied and Environmental Microbiology ◽

10.1128/aem.00892-17 ◽

2017 ◽

Vol 83 (16) ◽

Cited By ~ 11

Author(s):

Jasmine M. Bracher ◽

Erik de Hulster ◽

Charlotte C. Koster ◽

Marcel van den Broek ◽

Jean-Marc G. Daran ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Specific Growth Rate ◽

Specific Growth ◽

Baker’S Yeast ◽

Industrial Biotechnology ◽

Baker's Yeast ◽

Laboratory Evolution ◽

Content Type ◽

Study Laboratory

ABSTRACT Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h−1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h−1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h−1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h−1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

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Improving isobutanol productivity through adaptive laboratory evolution in Saccharomyces cerevisiae

10.21203/rs.2.19485/v1 ◽

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.

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Influence of organic acids and organochlorinated insecticides on metabolism of Saccharomyces cerevisiae

Zbornik Matice srpske za prirodne nauke ◽

10.2298/zmspn0508207p ◽

2005 ◽

pp. 207-215 ◽

Cited By ~ 1

Author(s):

Dusanka Pejin ◽

Vesna Vasic

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Sugar Beet ◽

Specific Growth Rate ◽

Glycogen Content ◽

Specific Growth ◽

Raw Materials ◽

Stress Factors ◽

Adaptive Mechanism ◽

Ribonucleic Acids

Saccharomyces cerevisiae is exposed to different stress factors during the production: osmotic, temperature, oxidative. The response to these stresses is the adaptive mechanism of cells. The raw materials Saccharomyces cerevisiae is produced from, contain metabolism products of present microorganisms and protective agents used during the growth of sugar beet for example the influence of acetic and butyric acid and organochlorinated insecticides, lindan and heptachlor, on the metabolism of Saccharomyces cerevisiae was investigated and presented in this work. The mentioned compounds affect negatively the specific growth rate, yield, content of proteins, phosphorus, total ribonucleic acids. These compounds influence the increase of trechalose and glycogen content in the Saccharomyces cerevisiae cells.

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Anaplerotic reactions active during growth of Saccharomyces cerevisiae on glycerol

FEMS Yeast Research ◽

10.1093/femsyr/foz086 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Joeline Xiberras ◽

Mathias Klein ◽

Celina Prosch ◽

Zahabiya Malubhoy ◽

Elke Nevoigt

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Specific Growth Rate ◽

Pyruvate Carboxylase ◽

Reference Strain ◽

Specific Growth ◽

Glyoxylate Cycle ◽

Maximum Specific Growth Rate ◽

The Glyoxylate Cycle ◽

Anaplerotic Reactions

ABSTRACT Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h−1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.

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Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00270-20 ◽

2020 ◽

Vol 86 (12) ◽

Cited By ~ 1

Author(s):

Anna K. Wronska ◽

Meinske P. Haak ◽

Ellen Geraats ◽

Eva Bruins Slot ◽

Marcel van den Broek ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Large Scale ◽

De Novo ◽

Laboratory Strain ◽

Optimal Growth ◽

Industrial Applications ◽

Fast Growth ◽

Growth Media ◽

Free Medium ◽

Content Type

ABSTRACT Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae. This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae. IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae. Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

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Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

Applied and Environmental Microbiology ◽

10.1128/aem.00056-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4145-4148 ◽

Cited By ~ 66

Author(s):

Takanori Awata ◽

Mamoru Oshiki ◽

Tomonori Kindaichi ◽

Noriatsu Ozaki ◽

Akiyoshi Ohashi ◽

...

Keyword(s):

Growth Rate ◽

Specific Growth ◽

Maximum Specific Growth Rate ◽

Ammonium Oxidation ◽

Lower Growth ◽

Content Type ◽

Physiological Characterization ◽

Phylogenetic Affiliation ◽

Lower Growth Rate

ABSTRACTThe phylogenetic affiliation and physiological characteristics (e.g.,Ksand maximum specific growth rate [μmax]) of an anaerobic ammonium oxidation (anammox) bacterium, “CandidatusScalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “CandidatusScalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

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Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium

Applied and Environmental Microbiology ◽

10.1128/aem.02246-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 17-30 ◽

Cited By ~ 138

Author(s):

Ryan A. LaCroix ◽

Troy E. Sandberg ◽

Edward J. O'Brien ◽

Jose Utrilla ◽

Ali Ebrahim ◽

...

Keyword(s):

Escherichia Coli ◽

Rapid Growth ◽

Scientific Discovery ◽

Maximum Growth ◽

Time Frame ◽

Adaptive Laboratory Evolution ◽

Genetic Changes ◽

Laboratory Evolution ◽

Content Type ◽

K 12

ABSTRACTAdaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated usingEscherichia coliK-12 MG1655 on glucose minimal media at 37°C. By keepingE. coliin constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription generpoB, an 82-bp deletion between the metabolicpyrEgene andrph, and an IS element between the DNA structural genehnsandtdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

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