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September 22, 2019  |  

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.© 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

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September 22, 2019  |  

Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris.

Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

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September 22, 2019  |  

Genome evolution across 1,011 Saccharomyces cerevisiae isolates.

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single ‘out-of-China’ origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.

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September 22, 2019  |  

The N6-adenine methylation in yeast genome profiled by single-molecule technology.

The most common and abundant DNA modification is 5-meth- ylcytosine (5mC), which has been well-established as an epigenetic mark regulating gene expression in eukaryotes (Jones, 2012). Another DNA modification N6-methyldeoxyadenosine (6mA), pre- viously reported as a widespread DNA methylation in prokaryotes, plays an important role in gene expression, DNA replication, DNA repair, cell cycle progression and host-pathogen interaction (Messer and Noyer-Weidner, 1988; Lu et al., 1994; Collier et al., 2007). The knowledge of 6mA in eukaryotes has been very limited until the recent development of high-throughput sequencing and high-sensitive mass spectrometry technologies, which have greatly contributed to the investigation of 6mA in fungi, animals and plants (Fu et al., 2015; Greer et al., 2015; Zhang et al., 2015; Koziol et al., 2016; Liu et al., 2016; Wu et al., 2016; Liang et al., 2017; Mondo et al., 2017). Recent studies revealed that 6mA abundance is vari- able, and it is relative higher in Chlamydomonas and early- diverging fungi species than other eukaryotes. The distribution pat- terns of 6mA and their functions are not quite conserved among or- ganisms. 6mA was found enriched near the transcription start sites (TSS) in Chlamydomonas (Fu et al., 2015) and at the repeats in Drosophila, Mus musculus and Danio rerio (Zhang et al., 2015; Liu et al., 2016; Wu et al., 2016), and commonly depleted from gene exons in Xenopus laevis and M. musculus (Koziol et al., 2016). In several species, 6mA was associated with transcriptionally active genes (Fu et al., 2015; Mondo et al., 2017), and it was also found correlated with gene silencing in mammalian embryonic stem cells (Wu et al., 2016).

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September 22, 2019  |  

Inferring the minimal genome of Mesoplasma florum by comparative genomics and transposon mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

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September 22, 2019  |  

High-oleate yeast oil without polyunsaturated fatty acids.

Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements.We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ?9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the ?12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with >?90% oleate content.High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

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September 22, 2019  |  

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

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September 22, 2019  |  

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

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September 22, 2019  |  

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

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September 22, 2019  |  

Whole-genome analysis of three yeast strains used for production of sherry-like wines revealed genetic traits specific to Flor yeasts.

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for biological wine aging. We have sequenced the genomes of three flor strains originated from different geographic regions and used for production of sherry-like wines in Russia. According to the obtained phylogeny of 118 yeast strains, flor strains form very tight cluster adjacent to the main wine clade. SNP analysis versus available genomes of wine and flor strains revealed 2,270 genetic variants in 1,337 loci specific to flor strains. Gene ontology analysis in combination with gene content evaluation revealed a complex landscape of possibly adaptive genetic changes in flor yeast, related to genes associated with cell morphology, mitotic cell cycle, ion homeostasis, DNA repair, carbohydrate metabolism, lipid metabolism, and cell wall biogenesis. Pangenomic analysis discovered the presence of several well-known “non-reference” loci of potential industrial importance. Events of gene loss included deletions of asparaginase genes, maltose utilization locus, and FRE-FIT locus involved in iron transport. The latter in combination with a flor-yeast-specific mutation in the Aft1 transcription factor gene is likely to be responsible for the discovered phenotype of increased iron sensitivity and improved iron uptake of analyzed strains. Expansion of the coding region of the FLO11 flocullin gene and alteration of the balance between members of the FLO gene family are likely to positively affect the well-known propensity of flor strains for velum formation. Our study provides new insights in the nature of genetic variation in flor yeast strains and demonstrates that different adaptive properties of flor yeast strains could have evolved through different mechanisms of genetic variation.

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September 22, 2019  |  

Genomic and probiotic characterization of SJP-SNU strain of Pichia kudriavzevii.

The yeast strain SJP-SNU was investigated as a probiotic and was characterized with respect to growth temperature, bile salt resistance, hydrogen sulfide reducing activity, intestinal survival ability and chicken embryo pathogenicity. In addition, we determined the complete genomic and mitochondrial sequences of SJP-SNU and conducted comparative genomics analyses. SJP-SNU grew rapidly at 37 °C and formed colonies on MacConkey agar containing bile salt. SJP-SNU reduced hydrogen sulfide produced by Salmonella serotype Enteritidis and, after being fed to 4-week-old chickens, could be isolated from cecal feces. SJP-SNU did not cause mortality in 10-day-old chicken embryos. From 13 initial contigs, 11 were finally assembled and represented 10 chromosomal sequences and 1 mitochondrial DNA sequence. Comparative genomic analyses revealed that SJP-SNU was a strain of Pichia kudriavzevii. Although SJP-SNU possesses pathogenicity-related genes, they showed very low amino acid sequence identities to those of Candida albicans. Furthermore, SJP-SNU possessed useful genes, such as phytases and cellulase. Thus, SJP-SNU is a useful yeast possessing the basic traits of a probiotic, and further studies to demonstrate its efficacy as a probiotic in the future may be warranted.

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September 22, 2019  |  

Long-read sequencing data analysis for yeasts.

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ~41 h to generate a complete and well-annotated genome from ~100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

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September 22, 2019  |  

Horizontal transfer and proliferation of Tsu4 in Saccharomyces paradoxus.

Recent evidence suggests that horizontal transfer plays a significant role in the evolution of of transposable elements (TEs) in eukaryotes. Many cases of horizontal TE transfer (HTT) been reported in animals and plants, however surprisingly few examples of HTT have been reported in fungi.Here I report evidence for a novel HTT event in fungi involving Tsu4 in Saccharomyces paradoxus based on (i) unexpectedly high similarity between Tsu4 elements in S. paradoxus and S. uvarum, (ii) a patchy distribution of Tsu4 in S. paradoxus and general absence from its sister species S. cerevisiae, and (iii) discordance between the phylogenetic history of Tsu4 sequences and species in the Saccharomyces sensu stricto group. Available data suggests the HTT event likely occurred somewhere in the Nearctic, Neotropic or Indo-Australian part of the S. paradoxus species range, and that a lineage related to S. uvarum or S. eubayanus was the likely donor species. The HTT event has led to massive proliferation of Tsu4 in the South American lineage of S. paradoxus, which exhibits partial reproductive isolation with other strains of this species because of multiple reciprocal translocations. Full-length Tsu4 elements are associated with both breakpoints of one of these reciprocal translocations.This work shows that comprehensive analysis of TE sequences in essentially-complete genome assemblies derived from long-read sequencing provides new opportunities to detect HTT events in fungi and other organisms. This work also provides support for the hypothesis that HTT and subsequent TE proliferation can induce genome rearrangements that contribute to post-zygotic isolation in yeast.

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September 22, 2019  |  

A graph-based approach to diploid genome assembly.

Constructing high-quality haplotype-resolved de novo assemblies of diploid genomes is important for revealing the full extent of structural variation and its role in health and disease. Current assembly approaches often collapse the two sequences into one haploid consensus sequence and, therefore, fail to capture the diploid nature of the organism under study. Thus, building an assembler capable of producing accurate and complete diploid assemblies, while being resource-efficient with respect to sequencing costs, is a key challenge to be addressed by the bioinformatics community.We present a novel graph-based approach to diploid assembly, which combines accurate Illumina data and long-read Pacific Biosciences (PacBio) data. We demonstrate the effectiveness of our method on a pseudo-diploid yeast genome and show that we require as little as 50× coverage Illumina data and 10× PacBio data to generate accurate and complete assemblies. Additionally, we show that our approach has the ability to detect and phase structural variants.https://github.com/whatshap/whatshap.Supplementary data are available at Bioinformatics online.

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