Publications by authors named "Jean-Marc Daran"

113 Publications

gEL DNA: A Cloning- and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing.

CRISPR J 2021 Apr 23. Epub 2021 Apr 23.

Department of Biotechnology, Delft University of Biotechnology, Delft, Netherlands.

Even for the genetically accessible yeast , the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of Cas9 or Cas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases. These gRNA expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in . Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex DNA editing. These DNA fragments can be custom ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR toolbox for large-scale strain construction programs in and promises to be relevant for other less genetically accessible yeast species.
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http://dx.doi.org/10.1089/crispr.2020.0028DOI Listing
April 2021

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae.

Metab Eng 2021 05 20;65:11-29. Epub 2021 Feb 20.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629, HZ, Delft, the Netherlands. Electronic address:

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.
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http://dx.doi.org/10.1016/j.ymben.2021.02.004DOI Listing
May 2021

A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae.

Nucleic Acids Res 2021 02;49(3):1769-1783

Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2627HZ Delft, The Netherlands.

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.
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http://dx.doi.org/10.1093/nar/gkaa1167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897487PMC
February 2021

Entering GATTACA: yeast genomes: analysis, insights and applications.

FEMS Yeast Res 2021 01;20(8)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, the Netherlands.

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http://dx.doi.org/10.1093/femsyr/foaa064DOI Listing
January 2021

Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in .

Front Genet 2020 3;11:518. Epub 2020 Jun 3.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

The lager-brewing yeast is a hybrid between and with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.
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http://dx.doi.org/10.3389/fgene.2020.00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283523PMC
June 2020

Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences.

ACS Synth Biol 2020 06 3;9(6):1361-1375. Epub 2020 Jun 3.

Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ Delft, The Netherlands.

The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast . The present study, using as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization (, glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in .
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http://dx.doi.org/10.1021/acssynbio.0c00045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309318PMC
June 2020

Exploring the abundance of oleate hydratases in the genus Rhodococcus-discovery of novel enzymes with complementary substrate scope.

Appl Microbiol Biotechnol 2020 Jul 1;104(13):5801-5812. Epub 2020 May 1.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD-dependent oxidation of 10-hydroxystearic acid.
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http://dx.doi.org/10.1007/s00253-020-10627-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306040PMC
July 2020

Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts.

Nat Commun 2020 05 1;11(1):2144. Epub 2020 May 1.

School of Microbiology, Environmental Research Institute and APC Microbiome Ireland, University College Cork, Cork, T12YN60, Ireland.

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.
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http://dx.doi.org/10.1038/s41467-020-16073-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195364PMC
May 2020

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

Appl Environ Microbiol 2020 06 2;86(12). Epub 2020 Jun 2.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

Quantitative physiological studies on 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 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, -aminobenzoic acid (ABA), 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 -inositol, nicotinic acid, or ABA. 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. Many strains of , a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, -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 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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http://dx.doi.org/10.1128/AEM.00388-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267190PMC
June 2020

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

Appl Environ Microbiol 2020 06 2;86(12). Epub 2020 Jun 2.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

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. 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 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 laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the () ortholog isolated from one of these biotin prototrophs, , enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of () in other laboratory and industrial strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase Bio1. In aerobic cultures on biotin-free medium, strains expressing Bio1 showed a decreased susceptibility to contamination by biotin-auxotrophic This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of 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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http://dx.doi.org/10.1128/AEM.00270-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267198PMC
June 2020

Vitamin requirements and biosynthesis in Saccharomyces cerevisiae.

Yeast 2020 04 6;37(4):283-304. Epub 2020 Feb 6.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term "vitamin" is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.
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http://dx.doi.org/10.1002/yea.3461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187267PMC
April 2020

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870.

BMC Genomics 2020 Jan 2;21(1). Epub 2020 Jan 2.

Protein Technologies, CSIR Biosciences, Meiring Naude Road, Brummeria, Pretoria, South Africa.

Background: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst.

Results: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci.

Conclusions: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.
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http://dx.doi.org/10.1186/s12864-019-6405-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941271PMC
January 2020

Chromosome level assembly and comparative genome analysis confirm lager-brewing yeasts originated from a single hybridization.

BMC Genomics 2019 Dec 2;20(1):916. Epub 2019 Dec 2.

Delft Bioinformatics Lab, Delft University of Technology, 2628, CD, Delft, The Netherlands.

Background: The lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S. eubayanus with extensive chromosome aneuploidy. S. pastorianus is subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity for S. cerevisiae chromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies of S. pastorianus strains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history.

Results: To fill this gap, we generated a chromosome-level genome assembly of the S. pastorianus strain CBS 1483 from Oxford Nanopore MinION DNA sequencing data and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history of S. pastorianus strains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between the S. cerevisiae subgenomes of Group 1 and 2 strains, and marked differences from sequenced S. cerevisiae strains.

Conclusions: Our findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygous S. cerevisiae strain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.
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http://dx.doi.org/10.1186/s12864-019-6263-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889557PMC
December 2019

Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae.

Metab Eng 2019 12 28;56:165-180. Epub 2019 Sep 28.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, the Netherlands. Electronic address:

The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.
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http://dx.doi.org/10.1016/j.ymben.2019.09.011DOI Listing
December 2019

Lager-brewing yeasts in the era of modern genetics.

FEMS Yeast Res 2019 11;19(7)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains.
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http://dx.doi.org/10.1093/femsyr/foz063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790113PMC
November 2019

Himalayan Genome Sequences Reveal Genetic Markers Explaining Heterotic Maltotriose Consumption by Saccharomyces pastorianus Hybrids.

Appl Environ Microbiol 2019 11 30;85(22). Epub 2019 Oct 30.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

strains are hybrids of and that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of genomes are being resolved, molecular mechanisms and evolutionary origins of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early × hybrids. To address this question, we generated a nearly complete genome assembly of Himalayan strains of the Holarctic subclade. This group of strains has been proposed to be the subgenome origin of current strains. The Himalayan genomes harbored several copies of an () α-oligoglucoside transporter gene with high sequence identity to genes encountered in Although Himalayan strains cannot grow on maltose and maltotriose, their maltose-hydrolase and and maltose transporter genes complemented the corresponding null mutants of Expression, in Himalayan of a functional maltose metabolism regulator gene () enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in is a result of heterosis was experimentally tested by constructing an × laboratory hybrid with a complement of maltose metabolism genes that resembles that of current strains. The ability of this hybrid to consume maltotriose in brewer's wort demonstrated regulatory cross talk between subgenomes and thereby validated this hypothesis. These results support experimentally the new postulated hypothesis on the evolutionary origin of an essential phenotype of lager brewing strains and valuable knowledge for industrial exploitation of laboratory-made -like hybrids., an × hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and colonized early lager brewing processes. Despite accumulation and analysis of genome sequencing data of parental genomes, the genetic blueprint of industrially relevant phenotypes remains unresolved. Assimilation of maltotriose, an abundant sugar in wort, has been postulated to be inherited from the parent. Here, we demonstrate that although Asian isolates harbor a functional maltotriose transporter gene, they are unable to grow on α-oligoglucosides, but expression of regulator () was sufficient to restore growth on trisaccharides. We hypothesized that the maltotriose phenotype results from regulatory interaction between maltose transcription activator and the promoter of We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains.
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http://dx.doi.org/10.1128/AEM.01516-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821976PMC
November 2019

Biological Parts for Synthetic Biology.

Front Bioeng Biotechnol 2019 7;7:97. Epub 2019 May 7.

School of Microbiology, Centre for Synthetic Biology and Biotechnology, Environmental Research Institute, APC Microbiome Institute, University College Cork, Cork, Ireland.

is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to . The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.
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http://dx.doi.org/10.3389/fbioe.2019.00097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515861PMC
May 2019

Phenotype-Independent Isolation of Interspecies Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting.

Front Microbiol 2019 26;10:871. Epub 2019 Apr 26.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

Interspecies hybrids of species are found in a variety of industrial environments and often outperform their parental strains in industrial fermentation processes. Interspecies hybridization is therefore increasingly considered as an approach for improvement and diversification of yeast strains for industrial application. However, current hybridization methods are limited by their reliance on pre-existing or introduced selectable phenotypes. This study presents a high-throughput phenotype-independent method for isolation of interspecies hybrids based on dual dye-staining and subsequent mating of two strains, followed by enrichment of double-stained hybrid cells from a mating population by fluorescence-activated cell sorting (FACS). Pilot experiments on intra-species mating of heterothallic haploid strains showed that 80% of sorted double-stained cells were hybrids. The protocol was further optimized by mating an haploid with homothallic spores with complementary selectable phenotypes. In crosses without selectable phenotype, using and haploids derived from laboratory as well as industrial strains, 10 to 15% of double-stained cells isolated by FACS were hybrids. When applied to rare mating, sorting of double-stained cells consistently resulted in about 600-fold enrichment of hybrid cells. Mating of dual-stained cells and FACS-based selection allows efficient enrichment of interspecies hybrids within a matter of days and without requiring selectable hybrid phenotypes, both for homothallic and heterothallic strains. This strategy should accelerate the isolation of laboratory-made hybrids, facilitate research into hybrid heterosis and offer new opportunities for non-GM industrial strain improvement and diversification.
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http://dx.doi.org/10.3389/fmicb.2019.00871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498416PMC
April 2019

Multiplex genome editing of microorganisms using CRISPR-Cas.

FEMS Microbiol Lett 2019 04;366(8)

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.
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http://dx.doi.org/10.1093/femsle/fnz086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522427PMC
April 2019

Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids.

J Agric Food Chem 2019 Dec 2;67(49):13478-13486. Epub 2019 May 2.

Laboratory of Food Chemistry , Wageningen University & Research , Bornse Weilanden 9 , 6708 WG Wageningen , Netherlands.

Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast . As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from in naringenin-producing yeast strains resulted in production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.
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http://dx.doi.org/10.1021/acs.jafc.9b01367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909231PMC
December 2019

Laboratory Evolution of a × Hybrid Under Simulated Lager-Brewing Conditions.

Front Genet 2019 29;10:242. Epub 2019 Mar 29.

Industrial Microbiology, Department of Biotechnology Delft, Delft University of Technology, Delft, Netherlands.

lager-brewing yeasts are domesticated hybrids of x that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of and was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid brewing strains. In contrast to the extensive aneuploidy of domesticated strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of × hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.
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http://dx.doi.org/10.3389/fgene.2019.00242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455053PMC
March 2019

In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation.

PLoS Genet 2019 04 4;15(4):e1007853. Epub 2019 Apr 4.

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands.

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.
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http://dx.doi.org/10.1371/journal.pgen.1007853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448828PMC
April 2019

The Genetic Makeup and Expression of the Glycolytic and Fermentative Pathways Are Highly Conserved Within the Genus.

Front Genet 2018 16;9:504. Epub 2018 Nov 16.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

The ability of the yeast to convert glucose, even in the presence of oxygen, via glycolysis and the fermentative pathway to ethanol has played an important role in its domestication. Despite the extensive knowledge on these pathways in , relatively little is known about their genetic makeup in other industrially relevant yeast species. In this study we explore the diversity of the glycolytic and fermentative pathways within the genus using , , and as paradigms. Sequencing data revealed a highly conserved genetic makeup of the glycolytic and fermentative pathways in the three species in terms of number of paralogous genes. Although promoter regions were less conserved between the three species as compared to coding sequences, binding sites for Rap1, Gcr1 and Abf1, main transcriptional regulators of glycolytic and fermentative genes, were highly conserved. Transcriptome profiling of these three strains grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs. Furthermore, transplantation of the promoters of major paralogs of and into demonstrated not only the transferability of these promoters, but also the similarity of their strength and response to various environmental stimuli. The relatively low homology of and promoters to their relatives makes them very attractive alternatives for strain construction in , thereby expanding the molecular toolbox.
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http://dx.doi.org/10.3389/fgene.2018.00504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250768PMC
November 2018

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

Nucleic Acids Res 2019 02;47(3):1362-1372

Department of Biotechnology, Delft University of Technology, Delft 2629HZ, The Netherlands.

Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.
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http://dx.doi.org/10.1093/nar/gky1216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379674PMC
February 2019

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains.

FEMS Yeast Res 2019 01;19(1)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, Delft 2629HZ, The Netherlands.

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.
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http://dx.doi.org/10.1093/femsyr/foy107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217715PMC
January 2019

Structural, Physiological and Regulatory Analysis of Maltose Transporter Genes in CBS 12357.

Front Microbiol 2018 10;9:1786. Epub 2018 Aug 10.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

lager brewing yeasts are domesticated hybrids of and cold-tolerant . To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the type strain CBS 12357/FM1318. To obtain complete sequences of the loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of in a maltose-transport-deficient strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only and , which shared 99.7% sequence identity, eliminated growth of CBS 12357 on maltose. Transcriptome analysis of CBS 12357 established that and , are poorly expressed in maltose-grown cultures, while and were expressed at much higher levels than and , indicating that only are responsible for maltose consumption in CBS 12357. These results represent a first genomic and physiological characterization of maltose transport in CBS 12357 and provides a valuable resource for further industrial exploitation of this yeast.
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http://dx.doi.org/10.3389/fmicb.2018.01786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097016PMC
August 2018

Selection of Pof Variants for the Construction of × Hybrids With Reduced 4-Vinyl Guaiacol Formation.

Front Microbiol 2018 27;9:1640. Epub 2018 Jul 27.

Department of Biotechnology, Delft University of Technology, Delft, Netherlands.

is an interspecies hybrid between and . The identification of the parental species of enabled the reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof (phenolic off-flavor). However, all known strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non-GMO approach to construct Pof variants derived from the parental strain CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof and Pof strains to cinnamic acid. The variant HTSE042 was selected and was confirmed not to produce 4-vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes whose deletion in a naïve strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof variant was crossed with a Pof partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct × hybrids with phenotypic characteristics of .
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http://dx.doi.org/10.3389/fmicb.2018.01640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6074607PMC
July 2018

Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains.

FEMS Yeast Res 2018 12;18(8)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

d-Glucose, d-xylose and l-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three 'specialist' Saccharomyces cerevisiae strains. A d-glucose- and l-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented d-xylose in the presence of 20 g L-1 of d-glucose and l-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose non-fermenting laboratory strain, rapidly and simultaneously converted d-glucose and l-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved 'generalist' strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1d-glucose, 10 g L-1d-xylose and 5 g L-1l-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.
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http://dx.doi.org/10.1093/femsyr/foy075DOI Listing
December 2018

Engineering de novo anthocyanin production in Saccharomyces cerevisiae.

Microb Cell Fact 2018 Jul 3;17(1):103. Epub 2018 Jul 3.

Wageningen Plant Research, Wageningen University & Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.

Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/g, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.

Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.
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http://dx.doi.org/10.1186/s12934-018-0951-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029064PMC
July 2018

Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive l-arabinose uptake.

FEMS Yeast Res 2018 09;18(6)

Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h-1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.
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http://dx.doi.org/10.1093/femsyr/foy062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044391PMC
September 2018