Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis

Verhoeven, Maarten D.; Lee, Misun; Kamoen, Lycka; Broek, Marcel van den; Janssen, Dick B.; Daran, Jean‐Marc; Maris, Antonius J. A. van; Pronk, Jack T.

doi:10.1038/srep46155

Cited by 65 publications

(107 citation statements)

References 70 publications

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“…Paired-end sequencing (126-bp reads) was performed on 350-bp insert libraries with an Illumina HiSeq 2500 sequencer (Baseclear BV, Leiden, The Netherlands) with a minimum sample size of 550 Mb, accounting for a coverage of approximately 45 times. Data mapping to the CEN.PK113-7D genome (22), data processing, and chromosome copy number variation determinations were done as described previously (65). Copy numbers of BIO1, BIO2, BIO3, BIO4, and BIO6 were estimated by comparing their read depths to the average read depths of the single-copy reference genes [YAL001C (TFC3), YBL015W (ACH1), YCL040W (GLK1), YDL029W (ARP2), YEL012W (UBC8), YER049W (TPA1), YBR196C (PGI1), YER178W (PDA1), YFL039C (ACT1), and YJL121C (RPE1)], processed with Pilon (66).…”

Section: Methodsmentioning

confidence: 99%

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

Bracher

Hulster

Koster

et al. 2017

Appl Environ Microbiol

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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 [] Ϸ 0.01 h Ϫ1 ). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates ( Յ 0.36 h Ϫ1 ) 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 ( ϭ 0.15 h Ϫ1 ). 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 Ϫ1 . 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.KEYWORDS Saccharomyces cerevisiae, adaptive laboratory evolution, biotin, prototrophy, reverse metabolic engineering, vitamin biosynthesis, whole-genome sequencing

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Section: Methodsmentioning

confidence: 99%

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

Bracher

Hulster

Koster

et al. 2017

Appl Environ Microbiol

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“…At low concentration, Mn 2+ was shown to be a stronger activator of Thi80 than Mg 2+ (58). In an ALE experiment with engineered xylose-fermenting assimilating S. cerevisiae , a non-sense mutation or deletion of PMR1 caused selectively and strongly increased intracellular concentrations of Mn 2+ , which was the preferred metal ion for the heterologously expressed Piromyces xylose isomerase (59). Although intracellular metal ion concentrations were not measured in the current study, the different phenotypes of a pmr1 Δ deletion strain (IMX1722) and a PMR1 S104Y strain (IMX1986) (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Data mapping was performed against the CEN.PK113-7D genome (22) where an extra chromosome containing the relative integration cassette was previously added. Data processing and chromosome copy number variation determinations were done as previously described (59, 69).…”

Section: Methodsmentioning

confidence: 99%

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

Perli

Moonen

Broek

et al. 2020

Preprint

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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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“…S. cerevisiae engineered with the XR-XDH pathway from S. stipitis, encoded by XYL1 and XYL2, have typically outperformed strains expressing bacterial XI. More recently, evolved strains of S. cerevisiae expressing fungal XI has led to faster growth rates and fewer byproducts (Zhou et al, 2012;Verhoeven et al, 2017). Kwak and Jin (2017) provide a review of engineering xylose fermentation in S. cerevisiae.…”

Section: Introductionmentioning

confidence: 99%

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

Correia

Khusnutdinova

et al. 2018

Preprint

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Xylose is the second most abundant sugar in lignocellulose and can be used as a feedstock for nextgeneration biofuels by industry. Saccharomyces cerevisiae, one of the main workhorses in biotechnology, is unable to metabolize xylose natively but has been engineered to ferment xylose to ethanol with the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Scheffersoymces stipitis. In the scientific literature, the yield and volumetric productivity of xylose fermentation to ethanol in engineered S. cerevisiae still lags S. stipitis, despite expressing of the same XR-XDH genes. These contrasting phenotypes can be due to differences in S. cerevisiae's redox metabolism that hinders xylose fermentation, differences in S. stipitis' redox metabolism that promotes xylose fermentation, or both. To help elucidate how S. stipitis ferments xylose, we used flux balance analysis to test various redox balancing mechanisms, reviewed published omics datasets, and studied the phylogeny of key genes in xylose fermentation. In vivo and in silico xylose fermentation cannot be reconciled without NADP phosphatase (NADPase) and NADH kinase. We identified eight candidate genes for NADPase. PHO3.2 was the sole candidate showing evidence of expression during xylose fermentation. Pho3.2p and Pho3p, a recent paralog, were purified and characterized for their substrate preferences. Only Pho3.2p was found to have NADPase activity. Both NADPase and NAD(P)H-dependent XR emerged from recent duplications in a common ancestor of Scheffersoymces and Spathaspora to enable efficient xylose fermentation to ethanol. This study demonstrates the advantages of using metabolic simulations, omics data, bioinformatics, and enzymology to reverse engineer metabolism. METHODSGenome-scale network reconstruction and analysis. The S. stipitis GENRE was obtained from a panfungal GENRE that combined the GENRE's of S. stipitis (Balagurunathan et al., 2012;Caspeta et al., 2012;Li, 2012;Liu et al., 2012), Schizosaccharomyces pombe (Sohn et al., 2012), Aspergillus niger (Andersen et al., 2008), Yarrowia lipolytica (Pan and Hua, 2012; Loira et al., 2012), Komagataella phaffii (Caspeta 3/28 et al., 2012), Kluyveromyces lactis (Dias et al., 2014), and S. cerevisiae (Heavner et al., 2013). Model simulations were carried out using COnstraints Based Reconstruction and Analysis for Python version 0.9.1 (COBRApy) (Ebrahim et al., 2013). The xylose uptake rate was set to 10 mmol · gDCW -1 · h -1 .The growth associated maintenance (GAM) and non-growth associated maintenance (NGAM) were set to 60 mmol · gDCW -1 and 0 mmol · gDCW -1 · h -1 , respectively. Flux variability analysis (FVA) was used to evaluate alternative optimum solutions (Mahadevan and Schilling, 2003). The exchange bounds for erythritol, ribitol, arabitol, sorbitol, and glycerol were all set to zero to simplify the solution space for polyols. The cofactor selectivities of NADH and NADPH were varied in a single XR reaction (Balagurunathan et al., 2012). XR solely driven by NADH or NADPH were blocked. The e...

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Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis

Cited by 65 publications

References 70 publications

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

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

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

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

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