Saturnispora sinensis sp. nov., a new ascomycetous yeast species from soil and rotten wood.

A yeast strain (CGMCC 2.6937T) belonging to the ascomycetous yeast genus Saturnispora was recently isolated from soil collected in Xinghuacun, Shanxi Province, PR China. The strain produces one or two ellipsoid or spherical ascospores in asci formed by the conjugation between a cell and its bud. Phylogenetic analyses of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene suggest that this strain is conspecific with strains NYNU 14639 isolated from rotten wood collected in Funiu Mountain, Henan province and ES13S05 from soil collected in Nantou County, Taiwan. The CGMCC 2.6937T group is most closely related to Saturnispora dispora and Saturnispora zaruensis. However, strain CGMCC 2.6937T differs from S. dispora by 17 (3.2 %, 13 substitutions and four gaps) and 77 (18.8 %, 52 substitutions and 25 gaps) mismatches, and from S. zaruensis by 15 (2.9 %, 12 substitutions and three gaps) and 64 (15.6 %, 44 substitutions and 20 gaps) mismatches, in the D1/D2 domain and ITS region, respectively. The results suggest that the CGMCC 2.6937T group represents an undescribed species in the genus Saturnispora, for which the name Saturnispora sinensis sp. nov. is proposed. The holotype strain is CGMCC 2.6937T.

Identification of dominant functional microbes that contribute to the characteristic aroma of Msalais, traditional wine fermented from boiled local grape juice in China.

Msalais is a traditional wine produced from naturally fermented boiled local grape juice in China. It has characteristic dried fruit and caramel odors, mainly attributed to aromatic compounds, such as furaneol and 5-methylfurfural. However, it is unclear how microbes involved in the natural fermentation of Msalais contribute to this characteristic aroma. Here, we analyzed the Msalais-fermenting microbes and aromatic compounds formed during natural Msalais fermentation by using high-throughput sequencing and gas chromatography-mass spectrometry, respectively. The analysis revealed that Saccharomyces cerevisiae, Kazachstania humilis, Lactobacillus plantarum, and Lactobacillus farraginis are the dominant and key functional species that produce high amounts of furaneol and 5-methylfurfural during Msalais fermentation. Of these, K. humilis and L. farraginis are rarely detected during regular wine fermentation. The identified functional species could be used to control typical aromatic characteristics of Msalais.

Yueomyces silvicola sp. nov., a novel ascomycetous yeast species unable to utilize ammonium, glutamate, and glutamine as sole nitrogen sources.

Five yeast strains isolated from tree bark and rotten wood collected in central and southwestern China, together with four Brazilian strains (three from soil and rotting wood collected in an Amazonian rainforest biome and one from Bromeliad collected in Alagoas state) and one Costa Rican strain isolated from a flower beetle, represent a new species closely related with Yueomyces sinensis in Saccharomycetaceae, as revealed by the 26S ribosomal RNA gene D1/D2 domain and the internal transcribed spacer region sequence analysis. The name Yueomyces silvicola sp. nov. is proposed for this new species with the holotype China General Microbiological Culture Collection Center 2.6469 (= Japan Collection of Microorganisms 34885). The new species exhibits a whole-genome average nucleotide identity value of 77.8% with Y. sinensis. The two Yueomyces species shared unique physiological characteristics of being unable to utilize ammonium and the majority of the amino acids, including glutamate and glutamine, as sole nitrogen sources. Among the 20 amino acids tested, only leucine and tyrosine can be utilized by the Yueomyces species. Genome sequence comparison showed that GAT1, which encodes a GATA family protein participating in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae, is absent in the Yueomyces species. However, the failure of the Yueomyces species to utilize ammonium, glutamate, and glutamine, which are generally preferred nitrogen sources for microorganisms, implies that more complicated alterations in the central nitrogen metabolism pathway might occur in the genus Yueomyces.

Microbial communities and their correlation with flavor compound formation during the mechanized production of light-flavor Baijiu.

Light-flavor Baijiu fermentation is a typical spontaneous solid-state fermentation process fueled by a variety of microorganisms. Mechanized processes have been increasingly employed in Baijiu production to replace traditional manual operation processes, however, the microbiological and physicochemical dynamics in mechanized processes remain largely unknown. Here, we investigated the microbial community succession and flavor compound formation during a whole mechanized fermentation process of light-flavor Baijiu using the conventional dilution plating method, PacBio single-molecule real-time (SMRT) sequencing and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The results showed that largely different fungal and bacterial communities were involved in the soaking and fermentation processes. A clear succession from Pantoea agglomerans to Bacillus (B.) smithii and B. coagulans in dominant bacterial species and from Cladosporium exasperatum to Saccharomyces cerevisiae and Lichtheimia ramosa in dominant fungal species occurred in the soaking processes. In the fermentation process, the most dominant bacterial species was shifted from Pantoea agglomerans to Lactobacillus (La.) acetotolerans and the most dominant fungal species were shifted from Lichtheimia ramose and Rhizopus arrhizus to Saccharomyces cerevisiae. The bacterial and fungal species positively associated with acidity and the formation of ethanol and different flavor compounds were specified. The microbial species exhibited strong co-occurrence or co-exclusion relationships were also identified. The results are helpful for the improvement of mechanized fermentation process of light-flavor Baijiu production.

Microbial Community Affects Daqu Quality and the Production of Ethanol and Flavor Compounds in Baijiu Fermentation.

Daqu is a traditional starter for Baijiu fermentation and is produced by spontaneous fermentation of ground and moistened barley or wheat. The quality of Daqu is traditionally evaluated based on physicochemical and subjective sensory parameters without microbiological analysis. Here, we compared the physicochemical characteristics of qualified (QD) and inferior (ID) Daqu, their microbial communities based on plate counting and PacBio SMRT sequencing of rRNA gene libraries, and their impacts on Baijiu fermentation. The results showed that the glucoamylase and α-amylase activities of QD were significantly higher than those of ID. The counts of yeasts and relative abundances of functional microbes, especially the amylolytic bacterium Bacillus licheniformis and fungi Saccharomycopsis fibuligera and Lichtheimia ramosa, were significantly higher in QD than in ID. The laboratory-scale Baijiu fermentation tests showed that the relative abundances of the amylolytic microbes were higher in the QD than the ID fermentation set, resulting in more efficient fermentation, as indicated by more weight loss and higher moisture content in the former. Consequently, more glycerol, acetic acid, ethanol, and other volatile compounds were produced in the QD than in the ID fermentation set. The results suggest that Daqu quality is determined by, and can be evaluated based on, its microbial community.

Highly diverged lineages of Saccharomyces paradoxus in temperate to subtropical climate zones in China.

The wild yeast Saccharomyces paradoxus has become a new model in ecology and evolutionary biology. Different lineages of S. paradoxus have been recognized across the world, but the distribution and genetic diversity of the species remain unknown in China, where the origin of its sibling species S. cerevisiae lies. In this study, we investigated the ecological and geographic distribution of S. paradoxus through an extensive field survey in China and performed population genomic analysis on a set of S. paradoxus strains, including 27 strains, representing different geographic and ecological origins within China, and 59 strains representing all the known lineages of the species recognized in the other regions of the world so far. We found two distinct lineages of S. paradoxus in China. The majority of the Chinese strains studied belong to the Far East lineage, and six strains belong to a novel highly diverged lineage. The distribution of these two lineages overlaps ecologically and geographically in temperate to subtropical climate zones in China. With the addition of the new China lineage, the Eurasian population of S. paradoxus exhibits higher genetic diversity than the American population. We observed more possible lineage-specific introgression events from the Eurasian lineages than from the American lineages. Our results expand the knowledge on ecology, genetic diversity, biogeography, and evolution of S. paradoxus.

Genetic diversity and population structure of the amylolytic yeast Saccharomycopsis fibuligera associated with Baijiu fermentation in China.

The amylolytic yeast Saccharomycopsis fibuligera is a predominant species in starters and the early fermentation stage of Chinese liquor (Baijiu). However, the genetic diversity of the species remains largely unknown. Here we sequenced the genomes of 97 S. fibuligera strains from different Chinese Baijiu companies. The genetic diversity and population structure of the strains were analyzed based on 1,133 orthologous genes and the whole genome single nucleotide polymorphisms (SNPs). Four main lineages were recognized. One lineage contains 60 Chinese strains which are exclusively homozygous with relatively small genome sizes (18.55-18.72 Mb) and low sequence diversity. The strains clustered in the other three lineages are heterozygous with larger genomes (21.85-23.72 Mb) and higher sequence diversity. The genomes of the homozygous strains showed nearly 100% coverage with the genome of the reference strain KPH12 and the sub-genome A of the hybrid strain KJJ81 at the above 98% sequence identity level. The genomes of the heterozygous strains showed nearly 80% coverage with both the sub-genome A and the whole genome of KJJ81, suggesting that the Chinese heterozygous strains are also hybrids with nearly 20% genomes from an unidentified source. Eighty-three genes were found to show significant copy number variation between different lineages. However, remarkable lineage specific variations in glucoamylase and α-amylase activities and growth profiles in different carbon sources and under different environmental conditions were not observed, though strains exhibiting relatively high glucoamylase activity were mainly found from the homozygous lineage.

Improved redox homeostasis owing to the up-regulation of one-carbon metabolism and related pathways is crucial for yeast heterosis at high temperature.

Heterosis or hybrid vigor is a common phenomenon in plants and animals; however, the molecular mechanisms underlying heterosis remain elusive, despite extensive studies on the phenomenon for more than a century. Here we constructed a large collection of F1 hybrids of Saccharomyces cerevisiae by spore-to-spore mating between homozygous wild strains of the species with different genetic distances and compared growth performance of the F1 hybrids with their parents. We found that heterosis was prevalent in the F1 hybrids at 40°C. A hump-shaped relationship between heterosis and parental genetic distance was observed. We then analyzed transcriptomes of selected heterotic and depressed F1 hybrids and their parents growing at 40°C and found that genes associated with one-carbon metabolism and related pathways were generally up-regulated in the heterotic F1 hybrids, leading to improved cellular redox homeostasis at high temperature. Consistently, genes related with DNA repair, stress responses, and ion homeostasis were generally down-regulated in the heterotic F1 hybrids. Furthermore, genes associated with protein quality control systems were also generally down-regulated in the heterotic F1 hybrids, suggesting a lower level of protein turnover and thus higher energy use efficiency in these strains. In contrast, the depressed F1 hybrids, which were limited in number and mostly shared a common aneuploid parental strain, showed a largely opposite gene expression pattern to the heterotic F1 hybrids. We provide new insights into molecular mechanisms underlying heterosis and thermotolerance of yeast and new clues for a better understanding of the molecular basis of heterosis in plants and animals.

Adaptive Gene Content and Allele Distribution Variations in the Wild and Domesticated Populations of Saccharomyces cerevisiae.

Recent studies on population genomics of Saccharomyces cerevisiae have substantially improved our understanding of the genetic diversity and domestication history of the yeast. However, the origin of the domesticated population of S. cerevisiae and the genomic changes responsible for ecological adaption of different populations and lineages remain to be fully revealed. Here we sequenced 64 African strains from various indigenous fermented foods and forests in different African countries and performed a population genomic analysis on them combined with a set of previously sequenced worldwide S. cerevisiae strains representing the maximum genetic diversity of the species documented so far. The result supports the previous observations that the wild and domesticated populations of S. cerevisiae are clearly separated and that the domesticated population diverges into two distinct groups associated with solid- and liquid-state fermentations from a single ancestor. African strains are mostly located in basal lineages of the two domesticated groups, implying a long domestication history of yeast in Africa. We identified genes that mainly or exclusively occur in specific groups or lineages and genes that exhibit evident group or lineage specific allele distribution patterns. Notably, we show that the homing endonuclease VDE is generally absent in the wild but commonly present in the domesticated lineages of S. cerevisiae. The genes with group specific allele distribution patterns are mostly enriched in functionally similar or related fundamental metabolism processes, including the evolutionary conserved TOR signaling pathway.

Two New Sexual Talaromyces Species Discovered in Estuary Soil in China.

In the survey of mycobiota of mudflats in China, two new sexually reproducing Talaromyces sect. Talaromyces species were discovered and studied using a polyphasic approach. These species are named here Talaromyces haitouensis (ex-type AS3.160101T) and Talaromyces zhenhaiensis (ex-type AS3.16102T). Morphologically, T. haitouensis is distinguished by moderate growth, green-yellow gymnothecia, orange-brown mycelium, and echinulate ellipsoidal ascospores. T. zhenhaiensis is characterized by fast growth, absence of sporulation, cream yellow to naphthalene yellow gymnothecia and mycelium, and smooth-walled ellipsoidal ascospores with one equatorial ridge. The two novelties are further confirmed by phylogenetic analyses based on either individual sequences of BenA, CaM, Rpb2, and ITS1-5.8S-ITS2 or the concatenated BenA-CaM-Rpb2 sequences.

Wickerhamomyces kurtzmanii sp. nov. An Ascomycetous Yeast Isolated From Crater Lake Water, Da Hinggan Ling Mountain, China.

One novel ascomycetous yeast strain TF5-16-2 was isolated from water samples of Tuofengling crater lake located in Da Hinggan Ling Mountain, in the Inner Mongolia province of China. Morphological, physiological characteristics, as well as phylogenetic analyses of D1/D2 domains of the large subunit rRNA (LSU), ITS region, small subunit rRNA (SSU), and elongation factor-1α (EF-1α) were performed and finally confirmed the phylogenetic placement of strain TF5-16-2 in the genus Wickerhamomyces. Sequences analysis revealed that strain TF5-16-2 differed from its most closely related phylogenetic neighbors 'Candida' silvicultrix CBS 6269T and Wickerhamomyces anomalus CBS 5759T by 8.0% (including 2.3% gaps), 8.5% (including 2.4% gaps) divergences in D1/D2 domains of LSU, and 11% (including 4.3% gaps) and 13% (including 4.4% gaps) divergences in ITS region, respectively. As the considerable sequence divergence and distinguishable physiological characteristics, strain TF5-16-2 was proposed as a new species of the genus Wickerhamomyces, with the name Wickerhamomyces kurtzmanii sp. nov. (holotype = CGMCC 2.5597, Mycobank number is MB829959).

Reverse Evolution of a Classic Gene Network in Yeast Offers a Competitive Advantage.

Glucose repression is a central regulatory system in yeast that ensures the utilization of carbon sources in a highly economical manner. The galactose (GAL) metabolism network is stringently regulated by glucose repression in yeast and has been a classic system for studying gene regulation. We show here that a Saccharomyces cerevisiae (S. cerevisiae) lineage in spontaneously fermented milk has swapped all its structural GAL genes (GAL2 and the GAL7-10-1 cluster) with early diverged versions through introgression. The rewired GAL network has abolished glucose repression and conversed from a strictly inducible to a constitutive system through polygenic changes in the regulatory components of the network, including a thymine (T) to cytosine (C) and a guanine (G) to adenine (A) transition in the upstream repressing sequence (URS) sites of GAL1 and GAL4, respectively, which impair Mig1p-mediated repression, loss of function of the repressor Gal80p through a T146I substitution in the protein, and subsequent futility of GAL3. Furthermore, the milk lineage of S. cerevisiae has achieved galactose-utilization rate elevation and galactose-over-glucose preference switch through the duplication of the introgressed GAL2 and the loss of function of the main glucose transporter genes HXT6 and HXT7. In addition, we demonstrate that GAL2 requires GAL7 or GAL10 for its expression, and Gal2p likely requires Gal1p for its transportation function in the milk lineage of S. cerevisiae. We show a clear case of reverse evolution of a classic gene network for ecological adaptation and provide new insights into the regulatory model of the canonical GAL network.

The origin and adaptive evolution of domesticated populations of yeast from Far East Asia.

The yeast Saccharomyces cerevisiae has been an essential component of human civilization because of its long global history of use in food and beverage fermentation. However, the diversity and evolutionary history of the domesticated populations of the yeast remain elusive. We show here that China/Far East Asia is likely the center of origin of the domesticated populations of the species. The domesticated populations form two major groups associated with solid- and liquid-state fermentation and appear to have originated from heterozygous ancestors, which were likely formed by outcrossing between diverse wild isolates primitively for adaptation to maltose-rich niches. We found consistent gene expansion and contraction in the whole domesticated population, as well as lineage-specific genome variations leading to adaptation to different environments. We show a nearly panoramic view of the diversity and life history of S. cerevisiae and provide new insights into the origin and evolution of the species.

Ballistosporomyces changbaiensis sp. nov. and Ballistosporomyces bomiensis sp. nov., two novel species isolated from shrub plant leaves.

Four strains, CB 266(T), CB 272, XZ 44D1(T) and XZ 49D2, isolated from shrub plant leaves in China were identified as two novel species of the genus Ballistosporomyces by the sequence analysis of the small subunit of ribosomal RNA (SSU rRNA), the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and internal transcribed spacer (ITS) + 5.8S rRNA region, and physiological comparisons. Ballistosporomyces changbaiensis sp. nov. (type strain CB 266(T) = CGMCC 2.02298(T) = CBS 10124(T), Mycobank number MB 815700) and Ballistosporomyces bomiensis sp. nov. (type strain XZ 44D1(T) = CGMCC 2.02661(T) = CBS 12512(T), Mycobank number MB 815701) are proposed to accommodate these two new species.

Evidence for a Far East Asian origin of lager beer yeast.

Lager-brewing arose in 15th century Bavaria [1] and is nowadays the most popular technique for alcoholic beverage production in the world. The technique is characterized by low temperature fermentation using the domesticated yeast Saccharomyces pastorianus (synonym S. carlsbergensis). It has been clear that the lager yeast is a hybrid with one portion of its genome having originated from S. cerevisiae ale yeast [2]. However, the source of the non-ale subgenome, which endows lager yeast with cold tolerance, had been a matter of debate [3]. Recently, a Patagonian origin hypothesis of lager yeast has been proposed based on the discovery of a new cryotolerant Saccharomyces species from Patagonian native forests of Argentina [4]. This yeast, named S. eubayanus, exhibited the closest known match (99.56%) to the non-ale portion of lager yeast and, thus, was believed to be its progenitor. However, we now show that this yeast species is likely native to the Tibetan Plateau. One of the Tibetan populations of the species exhibits closer affinity with lager yeast than the Patagonian population as inferred from population genetics and genome sequence analyses. We thus provide strong evidence for a Far East Asian origin hypothesis of lager yeast, which apparently corresponds better with geography and world trade history.

Naumovozyma baii sp. nov., an ascomycetous yeast species isolated from rotten wood in a tropical forest.

Two strains isolated from rotten wood were included in the Saccharomyces group based on morphological characteristics. However, rRNA gene sequence analyses (including the 18S rRNA gene, 26S rRNA gene D1/D2 domain and internal transcribed spacer region) indicated that these two strains represent a novel species of Naumovozyma, for which the name Naumovozyma baii sp. nov. is proposed (type strain: BW 22(T) = CGMCC 2.04520(T) = CBS 12642(T)). The MycoBank number of the new species is MB800484.

Udeniomyces kanasensis sp. nov., a ballistoconidium-forming yeast species in the Cystofilobasidiales.

In a survey of ballistoconidium-forming yeast diversity in the phyllosphere, five strains from wilting plant leaves collected from Kanas Nature Reserve in Xinjiang province, China were selected based on morphological comparison. These strains formed pinkish-white colonies and large bilaterally symmetrical ballistoconidia. Molecular phylogenetic analyses based on the 26S rRNA D1/D2 domain and ITS region sequences showed that these strains belonged to the Udeniomyces clade in the Cystofilobasidiales. They differ from the described Udeniomyces species significantly in the rRNA sequences as well as physiological criteria. Therefore, a new species Udeniomyces kanasensis sp. nov. (type strain XJ 6E2(T)=CGMCC 2.02627 (T)=CBS 12488 (T)) is proposed to accommodate these strains. The MycoBank number of the new species is MB 563659.