Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains.

Obligate anaerobic bacteria from the genus Pectinatus have been known to cause beer spoilage for over 40 years. Whole genome sequencing was performed on eleven beer spoilage strains (nine Pectinatus frisingensis, one Pectinatus cerevisiiphilus and one Pectinatus haikarae isolate), as well as two pickle spoilage species (Pectinatus brassicae MB591 and Pectinatus sottacetonis MB620) and the tolerance of all species to a range of environmental conditions was tested. Exploration of metabolic pathways for carbohydrates, amino acids and vitamins showed little difference between beer spoilage- and pickle spoilage-associated strains. However, genes for certain carbohydrate- and sulphur-containing amino acid-associated enzymes were only present in the beer spoilage group and genes for specific transporters and regulatory genes were uniquely found in the pickle spoilage group. Transporters for compatible solutes, only present in pickle-associated strains, likely explain their experimentally observed higher halotolerance compared to the beer spoilers. Genes involved in biofilm formation and ATP Binding Cassette (ABC) transporters potentially capable of exporting hop-derived antimicrobial compounds were found in all strains. All species grew in the presence of alcohol up to 5% alcohol by volume (ABV) and hops extract up to 80 ppm of iso-α-acids. Therefore, the species isolated from pickle processes may pose novel hazards in brewing.

Comparative genome analysis of the Lactobacillus brevis species.

BACKGROUND: Lactobacillus brevis is a member of the lactic acid bacteria (LAB), and strains of L. brevis have been isolated from silage, as well as from fermented cabbage and other fermented foods. However, this bacterium is also commonly associated with bacterial spoilage of beer. RESULTS: In the current study, complete genome sequences of six isolated L. brevis strains were determined. Five of these L. brevis strains were isolated from beer (three isolates) or the brewing environment (two isolates), and were characterized as beer-spoilers or non-beer spoilers, respectively, while the sixth isolate had previously been isolated from silage. The genomic features of 19 L. brevis strains, encompassing the six L. brevis strains described in this study and thirteen L. brevis strains for which complete genome sequences were available in public databases, were analyzed with particular attention to evolutionary aspects and adaptation to beer. CONCLUSIONS: Comparative genomic analysis highlighted evolution of the taxon allowing niche colonization, notably adaptation to the beer environment, with approximately 50 chromosomal genes acquired by L. brevis beer-spoiler strains representing approximately 2% of their total chromosomal genetic content. These genes primarily encode proteins that are putatively involved in oxidation-reduction reactions, transcription regulation or membrane transport, functions that may be crucial to survive the harsh conditions associated with beer. The study emphasized the role of plasmids in beer spoilage with a number of unique genes identified among L. brevis beer-spoiler strains.

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

Interspecies hybrids of Saccharomyces 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 Saccharomyces 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 S. cerevisiae strains showed that 80% of sorted double-stained cells were hybrids. The protocol was further optimized by mating an S. cerevisiae haploid with homothallic S. eubayanus spores with complementary selectable phenotypes. In crosses without selectable phenotype, using S. cerevisiae and S. eubayanus 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 Saccharomyces 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.

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.

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast.

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.

Genome annotation of a Saccharomyces sp. lager brewer's yeast.

The genome of lager brewer's yeast is a hybrid, with Saccharomyces eubayanus and Saccharomyces cerevisiae as sub-genomes. Due to their specific use in the beer industry, relatively little information is available. The genome of brewing yeast was sequenced and annotated in this study. We obtained a genome size of 22.7 Mbp that consisted of 133 scaffolds, with 65 scaffolds larger than 10 kbp. With respect to the annotation, 9939 genes were obtained, and when they were submitted to a local alignment, we found that 53.93% of these genes corresponded to S. cerevisiae, while another 42.86% originated from S. eubayanus. Our results confirm that our strain is a hybrid of at least two different genomes.

S. cerevisiae × S. eubayanus interspecific hybrid, the best of both worlds and beyond.

Saccharomyces pastorianus lager-brewing yeasts have descended from natural hybrids of S. cerevisiae and S. eubayanus. Their alloploidy has undoubtedly contributed to successful domestication and industrial exploitation. To understand the early events that have led to the predominance of S. pastorianus as lager-brewing yeast, an interspecific hybrid between S. cerevisiae and S. eubayanus was experimentally constructed. Alloploidy substantially improved the performance of the S. cerevisiae × S. eubayanus hybrid as compared to either parent regarding two cardinal features of brewing yeasts: tolerance to low temperature and oligosaccharide utilization. The hybrid's S. eubayanus subgenome conferred better growth rates and biomass yields at low temperature, both on glucose and on maltose. Conversely, the ability of the hybrid to consume maltotriose, which was absent in the S. eubayanus CBS12357 type strain, was inherited from its S. cerevisiae parent. The S. cerevisiae × S. eubayanus hybrid even outperformed its parents, a phenomenon known as transgression, suggesting that fast growth at low temperature and oligosaccharide utilization may have been key selective advantages of the natural hybrids in brewing environments. To enable sequence comparisons of the parental and hybrid strains, the genome of S. eubayanus CBS12357 type strain (Patagonian isolate) was resequenced, resulting in an improved publicly available sequence assembly.

De novo detection of copy number variation by co-assembly.

MOTIVATION: Comparing genomes of individual organisms using next-generation sequencing data is, until now, mostly performed using a reference genome. This is challenging when the reference is distant and introduces bias towards the exact sequence present in the reference. Recent improvements in both sequencing read length and efficiency of assembly algorithms have brought direct comparison of individual genomes by de novo assembly, rather than through a reference genome, within reach. RESULTS: Here, we develop and test an algorithm, named Magnolya, that uses a Poisson mixture model for copy number estimation of contigs assembled from sequencing data. We combine this with co-assembly to allow de novo detection of copy number variation (CNV) between two individual genomes, without mapping reads to a reference genome. In co-assembly, multiple sequencing samples are combined, generating a single contig graph with different traversal counts for the nodes and edges between the samples. In the resulting 'coloured' graph, the contigs have integer copy numbers; this negates the need to segment genomic regions based on depth of coverage, as required for mapping-based detection methods. Magnolya is then used to assign integer copy numbers to contigs, after which CNV probabilities are easily inferred. The copy number estimator and CNV detector perform well on simulated data. Application of the algorithms to hybrid yeast genomes showed allotriploid content from different origin in the wine yeast Y12, and extensive CNV in aneuploid brewing yeast genomes. Integer CNV was also accurately detected in a short-term laboratory-evolved yeast strain.

Engineering and analysis of a Saccharomyces cerevisiae strain that uses formaldehyde as an auxiliary substrate.

We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steady-state conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data.

This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.

Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures.

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production.

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.

Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast.

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects. An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C(2) independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S. cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).