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

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.     

Short bZIP homologue of sulfur regulator Met4 from Ogataea parapolymorpha does not depend on DNA-binding cofactors for activating genes in sulfur starvation

The acquisition of sulfur from environment and its assimilation is essential for fungal growth and activities. Here, we describe novel features of the regulatory network of sulfur metabolism in Ogataea parapolymorpha, a thermotolerant methylotrophic yeast with high resistance to harsh environmental conditions. A short bZIP protein (OpMet4p) of O. parapolymorpha, displaying the combined structural characteristics of yeast and filamentous fungal Met4 homologues, plays a key role as a master regulator of cell homeostasis during sulfur limitation, but also its function is required for the tolerance of various stresses. Domain swapping analysis, combined with deletion analysis of the regulatory domains and genes encoding OpCbf1p, OpMet28p, and OpMet32p, indicated that OpMet4p does not require the interaction with these DNA-binding cofactors to induce the expression of sulfur genes, unlike the Saccharomyces cerevisiae Met4p. ChIP analysis confirmed the notion that OpMet4p, which contains a canonical bZIP domain, can bind the target DNA in the absence of cofactors, similar to homologues in other filamentous fungi. Collectively, the identified unique features of the O. parapolymorpha regulatory network, as the first report on the sulfur regulation by a short yeast Met4 homologue, provide insights into conservation and divergence of the sulfur regulatory networks among diverse ascomycetous fungi.     

Isolation and characterization of thermotolerant yeasts for the production of second-generation bioethanol

The purpose of this study was to isolate, identify, and characterize the thermotolerant yeasts for use in high-temperature ethanol fermentation. Thermotolerant yeasts were isolated and screened from soil samples collected from the Mekong Delta, Vietnam, using the enrichment method. Classification and identification of the selected thermotolerant yeasts were performed using matrix-assisted laser desorption ionization/time-of-fight mass spectrometry (MALDI-TOF/MS) and nucleotide sequencing of the D1/D2 domain of the 26S rDNA and the internal transcribed spacer (ITS) 1 and 2 regions. The ethanol production by the selected thermotolerant yeast was carried out using pineapple waste hydrolysate (PWH) as feedstock. A total of 174 yeast isolates were obtained from 80 soil samples collected from 13 provinces in the Mekong Delta, Vietnam. Using MALDI-TOF/MS and nucleotide sequencing of the D1/D2 domain and the ITS 1 and 2 regions, six different yeast species were identified, including Meyerozyma caribbica, Saccharomyces cerevisiae, Candida tropicalis, Torulaspora globosa, Pichia manshurica, and Pichia kudriavzevii. Among the isolated thermotolerant yeasts, P. kudriavzevii CM4.2 displayed great potential for high-temperature ethanol fermentation. The maximum ethanol concentration (36.91 g/L) and volumetric ethanol productivity (4.10 g/L h) produced at 45 °C by P. kudriavzevii CM4.2 were achieved using PWH containing 103.08 g/L of total sugars as a feedstock. These findings clearly demonstrate that the newly isolated thermotolerant yeast P. kudriavzevii CM4.2 has a high potential for second-generation bioethanol production at high temperature.     

Electron transport chain in a thermotolerant yeast

Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ_m), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca²⁺ ions (Ca²⁺ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30^o, while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Maximum voluntary temperature of insect larvae reveals differences in their thermal biology

We investigate the thermoregulatory behaviors of larvae of four species of Drosophila (D. melanogaster, D. subobscura, D. pseudoobscura, and D. mojavensis), a thermotolerant strain of Drosophila melanogaster (T strain) known to differ in thermal biology, and two mutant stocks of D. melanogaster that have (as adults) defective thermoregulatory behavior. We describe and evaluate new techniques to measure two indices of maximum voluntary temperature of insect larvae. Both measures were highly repeatable within lines (species, strains, or mutants). One measure (temperature at which larvae stood upright) differed among lines consistent with expectations based on adult thermal ecology, suggesting that this measure will be useful measures of thermoregulatory set-points of larvae. The second measure (temperature of emergence from media) is less discriminatory.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Distinctive expression and cellular distribution of dopamine receptors in the pancreatic islets of rats

Activation of the dopamine (DA) D2 receptor inhibits glucose-stimulated insulin secretion in isolated rodent islets in vitro; however, no information is available regarding the cellular localization of DA receptors (DRs, including D1-D5 receptors) in pancreatic islets in situ. We investigate the protein expression and cellular localization of five types of DRs in pancreatic islets by means of Western blotting and double-labeling immunofluorescence in both normal control and alloxan-induced type 1 diabetes model (T1DM) rats. In control rats, D1 immunoreactivity (-IR) was distributed in the core of the islet and co-localized with insulin-IR, D2-IR was peripherally distributed and found only in somatostatin-immunoreactive cells and D5-IR was co-localized with glucagon-IR and pancreatic polypeptide-IR. No IR for either the D3 or D4 receptor was observed in rat islets. The protein level of the D1 receptor was reduced in T1DM rats (D1/D-glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 0.63?±?0.05 in control rats compared with 0.16?±?0.03 in T1DM rats, n?=?8, P?n?=?8, P?=?0.42) or the D5 receptor (D5/GAPDH, 0.50?±?0.04 compared with 0.47?±?0.04, n?=?8, P?=?0.58). The present study is the first clear demonstration of the protein expression and cellular localization of the D1, D2 and D5 receptors in rat pancreatic islets and provides crucial morphological evidence for further investigations of the underlying mechanism regarding the DA regulation of pancreatic endocrine function.     

Hybrid Selection of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Yeasts for Thermotolerance and Fermentation Activity

Molecular genetic screening of Saccharomyces yeasts, isolated from natural sources in the regions of the world with a hot climate (Africa, South America, Southeast and Central Asia) was used for the search of thermotolerant S. cerevisiae strains. Based on physiological tests, four strains were selected that could grow at high temperatures (42 and 43°C) and had good fermentation activity: 7962-4B, 3529-7B, 52922-4-1-1A- 1C, and 87-2421.1-2A. Hybrids of monosporic culture of distiller’s race XII (XII₇-2) with the thermotolerant strains were obtained. Unlike the strain XII₇-2, which is unable to grow at above 39°C, all hybrids showed good growth at 42°C. Two of the six hybrids analyzed, H2-1 (87-2421.1-2A × XII₇-2) and H3-2 (7962-4B × XII₇-2), showed higher fermentation activity than the parental strains. According to the results obtained, inter-strain hybridization is an efficient method of obtaining S. cerevisiae strains, which combine thermotolerance with high efficiency of alcoholic fermentation.     

Development of thermotolerant bacterial consortium,the basis for biopreparation for remediation of petroleum-contaminated soils and waters in hot climates

The consortium of thermotolerant petroleum-oxidizing bacteria containing strains Gordonia sp. 1D VKM Ac-2720 D, Rhodococcus sp. Par7 VKM Ac-2722 D, and R. pyridinivorans L5A-BSU VKM Ac-2721 for destruction of oil and oil products in hot climates was developed for the first time. The consortium was effective in soils and liquid media at temperature as high as 50°C, at salinity up to 7%, and soil moisture of about 10%. The efficiency of petroleum destruction for 21 days was 70 and 59% at 24 and 45°C, respectively. The consortium of thermotolerant petroleum-destructing strains could be used as basis for the biopreparation for remediation of petroleum-contaminated soils and waters in hot climates.     

Potential Role of Diploscapter sp. Strain LKC25, a Bacterivorous Nematode from Soil, as a Vector of Food-Borne Pathogenic Bacteria to Preharvest Fruits and Vegetables

Diploscapter, a thermotolerant, free-living soil bacterial-feeding nematode commonly found in compost, sewage, and agricultural soil in the United States, was studied to determine its potential role as a vehicle of Salmonella enterica serotype Poona, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes in contaminating preharvest fruits and vegetables. The ability of Diploscapter sp. strain LKC25 to survive on agar media, in cow manure, and in composted turkey manure and to be attracted to, ingest, and disperse food-borne pathogens inoculated into soil or a mixture of soil and composted turkey manure was investigated. Diploscapter sp. strain LKC25 survived and reproduced in lawns of S. enterica serotype Poona, E. coli O157:H7, and L. monocytogenes on agar media and in cow manure and composted turkey manure. Attraction of Diploscapter sp. strain LKC25 to colonies of pathogenic bacteria on tryptic soy agar within 10, 20, 30, and 60 min and 24 h was determined. At least 85% of the worms initially placed 0.5 to 1 cm away from bacterial colonies migrated to the colonies within 1 h. Within 24 h, ≥90% of the worms were embedded in colonies. The potential of Diploscapter sp. strain LKC25 to shed pathogenic bacteria after exposure to bacteria inoculated into soil or a mixture of soil and composted turkey manure was investigated. Results indicate that Diploscapter sp. strain LKC25 can shed pathogenic bacteria after exposure to pathogens in these milieus. They also demonstrate its potential to serve as a vector of food-borne pathogenic bacteria in soil, with or without amendment with compost, to the surface of preharvest fruits and vegetables in contact with soil.     

Kinetic study and mathematical modelling of killer and sensitive S. Cerevisiae strains growing in mixed culture

This paper presents a kinetic study of two yeasts growing in pure and mixed batch cultures. Two winemaking strains were used: S. cerevisiae K1 possessing the K2 killer character and S. cerevisiae 522D sensitive to the K2 killer toxin. Initially the kinetics of growth of the two strains were analysed in pure culture. In this case, the kinetic profiles of biomass production have shown that the growth rate of the K1 strain is slightly superior to the 522D strain. During the fermentation, the viability for both populations was higher than 90%. Fermentations in mixed culture with an initial percentage in killer strain of 5 and 10% with respect to the total population were carried out. The results showed a more important decrease in the percentage of total viable yeasts when the initial concentration of killer yeast increased. However, the kinetic profiles of total biomass (killer plus sensitive yeasts) were very similar for both fermentations. A mathematical model was proposed to simulate the microbial growth of the killer and sensitive strain developing in pure and mixed cultures. This mathematical model consists in three main reactions: the evolution of the killer toxin in the culture medium, the duplication and the mortality rates for each microbial population. The results of the simulation appeared in agreement with the experimental data.     

Improvement of thermotolerance in Lachancea thermotolerans using a bacterial selection pressure

The use of thermotolerant yeast strains is an important attribute for a cost-effective high temperature biofermentation processes. However, the availability of thermotolerant yeast strains remains a major challenge. Isolation of temperature resistant strains from extreme environments or the improvements of current strains are two major strategies known to date. We hypothesised that bacteria are potential “hurdles” in the life cycle of yeasts, which could influence the evolution of extreme phenotypes, such as thermotolerance. We subjected a wild-type yeast, Lachancea thermotolerans to six species of bacteria sequentially for several generations. After coevolution, we observed that three replicate lines of yeasts grown in the presence of bacteria grew up to 37 °C whereas the controls run in parallel without bacteria could only grow poorly at 35 °C retaining the ancestral mesophilic trait. In addition to improvement of thermotolerance, our results show that the fermentative ability was also elevated, making the strains more ideal for the alcoholic fermentation process because the overall productivity and ethanol titers per unit volume of substrate consumed during the fermentation process was increased. Our unique method is attractive for the development of thermotolerant strains or to augment the available strain development approaches for high temperature industrial biofermentation.

     

Screening and genetic identification of acidic and neutral protease-producing yeasts strains by 26S rRNA gene sequencing

Protease enzymes (proteases), particularly those produced by microorganisms, play very important roles in industry, due to their diverse applications. Considering the richness of microbial diversity in nature, a good chance always exists that proteases more suitable, with better properties for commercial application, may be discovered while screening novel microorganisms from local environments. In this study, 94 yeasts were isolated from different natural sources collected from the Abha region, Kingdom of Saudi Arabia, to determine extracellular protease production and activity. Among them, 23 isolates (24.46%) showed protease activity using a casein hydrolysis test. Of these, five isolates (21.74%) were selected and identified as the best protease producers by exhibiting the largest clearance zones around colonies. A 26S rRNA gene D1/D2 domain sequence alignment, comparison, and phylogenetic analysis of our study yeasts to published D1/D2 domain rRNA gene sequences from GenBank, identifies the isolates as Rhodotorula mucilaginosa KKU-M12c, Cryptococcus albidus KKU-M13c, Pichia membranifaciens KKU-M18c, Hanseniaspora uvarum KKU-M19c, and Candida californica KKU-M20c. The influence of varying pH (4.0–9.0) on the yield and activity of the proteases was investigated using 0.5% (w/v) casein as a substrate, to detect optimum pH values for yeast extracellular protease production. Enzyme activity was measured using qualitative and quantitative assays. Results show all of the study yeasts secreting protease enzyme at all tested pH levels, with the exception of pH 9.0. This indicates that none of the five yeasts are alkaline protease producers. Maximum protease activity (187 U/mL) was observed in strain H. uvarum KKU-M19c at pH 6.0 (only), indicating that strain KKU-M19c only produces neutral protease. The other four yeast isolates, R. mucilaginosa KKU-M12c, C. albidus KKU-M13c, P. membranifaciens KKU-M18c, and C. californica KKU-M20c, produced both acidic (at pH 4.0) and neutral (at pH 6.0 and 7.0) proteases. Strain C. californica KKU-M20c was found to be the best acidic and neutral protease producer (138 U/mL at pH 4.0, and 185 U/mL at pH 7.0). This is the first report of the discovery and isolation of local, powerful yeasts producing acidic and neutral protease enzymes from the Abha region, Kingdom of Saudi Arabia.     

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of “Rio Grande do Sul” (RS) accounts for 90% of Brazilian wines. The state of “Santa Catarina” (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.     

Detection of yeast species also occurring in substrates associated with animals and identification of a novel dimorphic species in Verbascum flowers from Georgia

The molecular taxonomic analysis of yeasts isolated from Verbascum flowers collected in central Georgia identified strains that could be assigned to the species Cryptococcus adeliensis, Cryptococcus magnus and Moniliella megachiliensis detected previously also in substrates associated with insects and other animals and a hitherto undescribed species for which the name Candida verbasci is proposed. The new species forms slightly pink colonies, propagates by mostly unipolar budding, forms invasive pseudomycelium, and the sequences of its D1/D2 LSU rRNA genes and ITS1-5.8S-ITS2 regions indicate close phylogenetic relationship with a group of species that form a cluster basal to the Candida albicans/Lodderomyces elongisporus clade. The type strain is 11-1055^T. It has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 12699^T, the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.02048^T and the Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 29-185-1^T. The GenBank accession numbers for nucleotide sequences of the C. verbasci type strain are: JX515981 (D1/D2 domain of the 26S rRNA gene) and JX515982 (ITS1-5.8S-ITS2). Mycobank: MB 801391.