Thermotolerant Strain Research Articles

Adaptation to Bleaching: Are Thermotolerant Symbiodiniaceae Strains More Successful Than Other Strains Under Elevated Temperatures in a Model Symbiotic Cnidarian?

The ability of some symbiotic cnidarians to resist and better withstand stress factors that cause bleaching is a trait that is receiving increased attention. The adaptive bleaching hypothesis postulates that cnidarians that can form a stable symbiosis with thermotolerant Symbiodiniaceae strains may cope better with increasing seawater temperatures. We used polyps of the scyphozoan, Cassiopea xamachana, as a model system to test symbiosis success under heat stress. We sought to determine: (1) if aposymbiotic C. xamachana polyps could establish and maintain a symbiosis with both native and non-native strains of Symbiodiniaceae that all exhibit different tolerances to heat, (2) whether polyps with these newly acquired Symbiodiniaceae strains would strobilate (produce ephyra), and (3) if thermally tolerant Symbiodiniaceae strains that established and maintained a symbiosis exhibited greater success in response to heat stress (even if they are not naturally occurring in Cassiopea). Following recolonization of aposymbiotic C. xamachana polyps with different strains, we found that: (1) strains Smic, Stri, Slin, and Spil all established a stable symbiosis that promoted strobilation and (2) strains Bmin1 and Bmin2 did not establish a stable symbiosis and strobilation did not occur. Strains Smic, Stri, Slin, and Spil were used in a subsequent bleaching experiment; each of the strains was introduced to a subset of aposymbiotic polyps and once polyp tissues were saturated with symbionts they were subjected to elevated temperatures - 32°C and 34°C - for 2 weeks. Our findings indicate that, in general, pairings of polyps with Symbiodiniaceae strains that are native to Cassiopea (Stri and Smic) performed better than a non-native strain (Slin) even though this strain has a high thermotolerance. This suggests a degree of partner specificity that may limit the adaptive potential of certain cnidarians to increased ocean warming. We also observed that the free-living, non-native thermotolerant strain Spil was relatively successful in resisting bleaching during experimental trials. This suggests that free-living Symbiodiniaceae may provide a supply of potentially “new” thermotolerant strains to cnidarians following a bleaching event.

Marine bacteria associated with shallow hydrothermal systems in the Gulf of California with the capacity to produce biofilm inhibiting compounds.

Shallow hydrothermal systems are extreme environments. The sediments and fluids emitted from the vents present unusual physical and chemical conditions compared to other marine areas, which promotes unique biodiversity that has been of great interest for biotechnology for some years. In this work, a bioprospective study was carried out to evaluate the capacity of bacteria associated with shallow hydrothermal vents to produce biofilm-inhibiting compounds. Degradation assays of N-acyl homoserine lactone (AHL) autoinducers (C6HSL) involved in the quorum sensing process were carried out on 161 strains of bacteria isolated from three shallow hydrothermal systems located in Baja California Sur (BCS), Mexico. The biosensor Chromobacterium violaceum CV026 was used. Twenty-three strains showed activity, and organic extracts were obtained with ethyl acetate. The potential of the extracts to inhibit the formation of biofilms was tested against two human pathogenic strains (Pseudomonas aeruginosa PAO1 and Aeromonas caviae ScH3), a shrimp pathogen (Vibrio parahaemolyticus M8), and two marine strains identified as producing biofilms on submerged surfaces (Virgibacillus sp C29 and Vibrio alginolyticus C96). The results showed that Vibrio alginolyticus and Brevibacillus thermoruber, as well as some thermotolerant strains (mostly Bacillus), produce compounds that inhibit bacterial biofilms (B. licheniformis, B. paralicheniformis, B. firmus, B. oceanizedimenis, B. aerius and B. sonorensis).

Nutrient removal from swine wastewater using a thermotolerant strain of Chlorella sp. grown under outdoor conditions

AbstractBACKGROUNDRecently, wastewater effluents from animal farms have been treated using microalgae for the removal of organic and inorganic compounds. Swine wastewater (SW) and wastewater treated by anaerobic digestion (TW) were used as culture media for a wild strain of microalga, Chlorella sp. (Chlorophyta), previously isolated for our research group. This was cultivated under laboratory conditions in a batch culture, and then inoculated in a cascade thin‐layer reactor under outdoor conditions. The kinetic behavior, nutrient removal (COD, NH4+‐N, NO3−‐N and PO34−‐P) efficiency and biochemical composition of the microalga biomass were evaluated.RESULTSSW supported the highest growth (339 × 106 cells mL−1 and μ = 0.42 d−1) and the maximal removal of COD (91%), nitrates (87%), ammonium (45%) and phosphate (70%) in a batch culture. Whereas, in continuous cultivation, an adequate operation was achieved at temperatures up to 42 °C, with removals greater than 80% of COD, 70% of nitrates, 90% of ammonium and 60% of phosphates. Also, the produced biomass achieved the highest content of proteins (386 mg g−1 dry weight), carbohydrates (291.49 mg g−1 dry weight) and saturated and monounsaturated fatty acids, reaching 74.76% of total fatty acids.CONCLUSIONSThe SW medium showed greater growth of Chlorella than TW using the outdoor thin‐layer cascade reactor. This is an opportunity to use wastewater as nutrient to obtain biomass enriched in compounds with various possibilities for application. © 2019 Society of Chemical Industry

Ethanol production from banana peels at high pretreated substrate loading: comparison of two operational strategies

The potential of banana peels (BPs) as a source of fermentable sugars for ethanol production was evaluated using two yeasts: Saccharomyces cerevisiae commercial strain and Kluyveromyces marxianus thermotolerant strain isolated from Agave honey. Different process configurations: simultaneous saccharification and fermentation (SSF), as well as presaccharification and simultaneous saccharification and fermentation (PSSF) at high solid loading (up to 25% w/w), were studied. Similar ethanol production 13 g/L and 11 g/L was achieved at 10–12 h on SSF for S. cerevisiae and K. marxianus at 35 and 41 °C, respectively, with 10% (w/w) BP. The highest ethanol concentration (32.6 g/L) was reached on PSSF process (at 64 h) using commercial S. cerevisiae strain with 25% (w/v) BP, while ethanol production was inhibited in the case of K. marxianus under same assay conditions. BPs are a suitable source for second-generation ethanol production. The advantage of S. cerevisiae in the PSSF in comparison with K. marxianus was demonstrated. The present study demonstrated that BPs are the promising feedstock for ethanol production by PSSF.

Analysis of genome sequence and trehalose lipid production peculiarities of the thermotolerant Gordonia strain.

Gordoniae are one of the most promising hydrocarbon-oxidizing actinobacteria. Here we present the genome sequence analysis of thermotolerant strain Gordonia sp. 1D isolated from oil-refinery soil. It is capable of alkane consumption and biosurfactant production at temperatures of up to 50°C. Gordonia sp. 1D demonstrates maximum biosurfactant production when grown on hexadecane, and at 40°C it was slightly higher than at 27°C: 35 and 39 mN/m, respectively. For the first time, it was experimentally confirmed that the carbohydrate component of extracellular biosurfactants produced by strain 1D is trehalose. In addition, genes for the production of trehalose lipid biosurfactants were identified. The genetic determinants for two different pathways for trehalose synthesis were found. The strain carries genes otsA and otsB involved in de novo trehalose biosynthesis. Moreover, the genes treY and treZ responsible for trehalose biosynthesis from maltooligosaccharides and starch or glycogen were identified.

Study of As-resistant bacteria from Nadia, India and a survey of two As resistance-related proteins.

The present investigation deals with the characterisation of three As-resistant bacteria, Bacillus aryabhattai strain VPS1, Bacillus licheniformis strain VPS6 and Sporosarcina thermotolerans strain VPS7 isolated from the rhizosphere of a contaminated paddy field in Chakdaha, Nadia, West Bengal, India. Two strains, VPS6 and VPS7 showed ureolytic activity, which can be used for microbial-induced calcite precipitation of As as a bioremediation option. However, As reduction and oxidation capacities were not reported in any of these bacteria. A phylogenetic tree of 16S ribosomal RNA gene sequences was constructed for all three bacterial isolates, including different species of As-resistant Bacillus and Sporosarcina. Furthermore, literature survey and genome mining were employed to explore the diversity of As resistance-related proteins, arsenite S-adenosylmethyltransferase (ArsM) and arsenical pump membrane protein (ArsB) among different bacteria, and the phylogenetic relatedness was studied to understand the distribution and evolution of their amino acid sequences. ArsB was predominantly present in a wide variety of bacteria (347 taxa); however, ArsM was reported in comparatively fewer isolates (109 taxa). There were a total of 60 similar taxa that contained both ArsM and ArsB. Both proteins were most abundantly present in phylum Proteobacteria. Overall, this investigation enumerates As-resistant bacteria to understand the As metabolism in the environment, and the phylogenetic analysis of As resistance-related proteins helps in understanding the functional relationship in different bacteria for their role in As mobility in the environment.

Alternative Photosynthetic Electron Transfers and Bleaching Phenotypes Upon Acute Heat Stress in Symbiodinium and Breviolum spp. (Symbiodiniaceae) in Culture

The breakdown of the symbiosis between cnidarians and Symbiodiniaceae often occurs upon periods of elevated sea surface temperature and gives rise to bleaching events that affect coral reefs worldwide. In the prevailing model explaining the cellular mechanisms of bleaching, an impairment of photosynthesis would be responsible for light-dependent generation of toxic reactive oxygen species and ultimately for death of symbionts and/or host cells. In some Symbiodiniaceae species, alternative photosynthetic electron flows (AEF) have been documented to occur upon a shift to high temperature, possibly contributing to photoprotection and to the balance of energetic ratio between photoproduced ATP and NADPH. By using a combination of in vivo spectrofluorimetric and oximetric techniques, we studied the capacity for electron rerouting towards oxygen and cyclic electron flow around photosystem I in nine Symbiodiniaceae species belonging to Symbiodinium and Breviolum genera upon a shift from 25°C to 33°C. CEF capacity was determined as the kinetic of PSI primary donor P700 re-reduction in the presence of DCMU, a PSII inhibitor. An active oxygen uptake in light was estimated by comparing net oxygen evolution and relatove electron transport rate of PSII at different light intensities. Among strains that showed elevated capacity for both AEF, some were thermotolerant while others were thermosensitive. Conversely, in some thermotolerant strains, capacities for these AEF were low upon heat stress. Our results thus indicate that a high CEF capacity and/or a high ability to reroute electrons towards O2 are not sufficient to confer thermal resistance of isolated Symbiodiniaceae.

Unravelling efficient applications of agriculturally important microorganisms for alleviation of induced inter-cellular oxidative stress in crops

Abiotic stresses like high temperature, cold, freezing, drought, salinity, flooding or oxidizing agents cause significant loss in the crop yield and quality. Abiotic stresses cause reactive oxygen species (ROS) production such as singlet oxygen (1O2), hydrogen peroxide (H2O2), superoxide radical (O2•−), hydroxyl radical (OH-), etc., that leads to a significant reduction of crop yield. A major source of ROS production in plants through aerobic metabolism is chloroplast, mitochondria, and peroxisome. The tripartite interactions involving Trichoderma- Phytopathogen-Host have received less attention in contrast to the plant–antagonist, plant–pathogen or pathogen–antagonist interactions. This article explores the possibilities of employing thermotolerant strains of agriculturally important microorganisms (AIMs) for alleviating the oxidative stress induced due heat stress in crops by modulating oxidative and defense network of the host.

Hiperproducción de carbometilcelulasa por Thermomyces dupontii utilizando mutagénesis física y química

Utilization of cellulases as substitute of chemical process gained huge momentum in the field of biotechnology  Now there is dire need to find out un explore reveres of fungi possessing greater potential for efficient cellulase production.  This boosted isolation of novel thermo tolerant fungal strains capable of producing the targeted product. In this investigation 70 thermophilic cellulolytic fungal strains were isolated. All the strains were screened via submerged fermentation. The strain showing highest CMCase activity was identified by conventional method i.e.  based on morphology and microscopic features and confirmed by 18S rDNA gene sequencing, using specific ITS primers. The modified CTAB method was used for rapid extraction of DNA from thermo tolerant strain.  The selected strain subsequently subjected to sequencing and phylogenetic analysis. The result indicates the selected strain was found to be T. dupontii. For strain improvement the T. dupontii was subjected to random mutagenesis by using (UV) irradiation and EMS treatment. Out of 40 screened mutant T. dupontii EMS 15 had 2.4 fold more yield (21.8 U/ml/min) as compared to parental strain. The Five fermentation media were also screened. The Medium3 gave higher titer of cellulase activity (29.3 U/ml/min) and found to be the best medium.

Investigation of Anti-archaeal Activities of Some Commercial Essential Oils on Extremely Halophilic Archaea

In this study, the anti-archaeal activity of thyme (Thymus spp.), mint (Mentha piperita), black garlic (Allium sativum) commercial essential oils were investigated on the extremely halophilic archaea. Halococcus morrhuae strain 123, Haloarchaeon 129, Halorubrum sp. 140, Natrinema pallidum strain 153, Natrialba aegyptia strain 213, Haloterrigena thermotolerans strain 415, Halococcus thailandensis strain 514 and Halobacterium noricense strain 714 were used as extremely halophilic archaea strains. SW25 broth - agar media were used for the growth of cultures. Commercial essential oils were tested at 1/10, 1/50 and 1/100 concentrations. Agar disc diffusion method was applied for antimicrobial activity. Bacitracin (B10), Novobiocin (NV5), Penicillin G (P10), Fusidic acid (FA10) antibiotic discs and Haloferax sp. HSC4 extremely halophilic archaea type culture were used as control. Commercial thyme, mint and black garlic essential oils tested in the study were found to have anti-archeal activity against all halophilic archaeal strains, 5 strains and 3 strains, respectively. According to our results, the highest inhibition zone diameters were obtained from thyme essential oil. Keywords: Thyme, Mint, Black Garlic, Essential Oil, Archaea DOI: 10.7176/JSTR/5-8-07

Oxidative stress resistance during dehydration of three non-Saccharomyces wine yeast strains.

The development of new fermented foods and beverages requires more and more the use of new dehydrated yeasts species. In this context, the non-Saccharomyces (NS) yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans are developed especially in winemaking as co-culture in the fermentation of the must or for the must bioprotection. However, during formulation-dehydration the yeast cells are exposed to several stresses that reduce cellular activity. Among these, the oxidative stress induced by the stabilization processes has been described as one of the main causes of cell death. In this study, we analyzed the effects of growth medium associated with two dehydration kinetics on the accumulation of reactive oxygen species (ROS) and lipid peroxidation levels. The cultivability of tested yeast strains was dependent on growth and dehydration conditions. The L. thermotolerans strain was the most sensible to dehydration when growing in nutrient-poor media, which was characterized by high levels of ROS, lipid peroxidation and reduced cultivability. In contrast, this yeast was able to restore its cultivability when growing in nutrient-rich medium before dehydration. The other NS yeast strains acquired resistance to oxidative stress similarly but in a growth-dehydration condition less dependent manner. These results showed that modulation of growing medium composition is a simple way to improve resistance to oxidative attack imposed by dehydration in NS yeasts. This was the first time that multiple quantitative and qualitative indicators of oxidative stress response in these three NS yeast strains were explored.

Effect of Sequential Acclimation to Various Carbon Sources on the Proteome of Acetobacter senegalensis LMG 23690T and Its Tolerance to Downstream Process Stresses.

Acetic acid bacteria are very vulnerable to environmental changes; hence, they should get acclimated to different kinds of stresses when they undergo downstream processing. In the present study, Acetobacter senegalensis LMG 23690T, a thermo-tolerant strain, was acclimated sequentially to different carbon sources including glucose (condition Glc), a mixture of glucose and ethanol (condition EtOH) and a mixture of glucose and acetic acid (condition GlcAA). Then, the effects of acclimation on the cell proteome profiles and some phenotypic characteristics such as growth in culture medium containing ethanol, and tolerance to freeze-drying process were evaluated. Based on the obtained results, despite the cells acclimated to Glc or EtOH conditions, 86% of acclimated cells to GlcAA condition were culturable and resumed growth with a short lag phase in a culture medium containing ethanol and acetic acid. Interestingly, if A. senegalensis LMG 23690T had been acclimated to condition GlcAA, 92% of cells exhibited active cellular dehydrogenases, and 59% of cells were culturable after freeze-drying process. Proteome profiles comparison by 2D-DiGE and MS analysis, revealed distinct physiological status between cells exposed to different acclimation treatments, possibly explaining the resulting diversity in phenotypic characteristics. Results of proteome analysis by 2D-DiGE also showed similarities between the differentially expressed proteins of acclimated cells to EtOH condition and the proteome of acclimated cells to GlcAA condition. Most of the differentially regulated proteins are involved in metabolism, folding, sorting, and degradation processes. In conclusion, acclimation under appropriate sub-lethal conditions can be used as a method to improve cell phenotypic characteristics such as viability, growth under certain conditions, and tolerance to downstream processes.

QTL analysis reveals genomic variants linked to high-temperature fermentation performance in the industrial yeast

BackgroundHigh-temperature fermentation is desirable for the industrial production of ethanol, which requires thermotolerant yeast strains. However, yeast thermotolerance is a complicated quantitative trait. The understanding of genetic basis behind high-temperature fermentation performance is still limited. Quantitative trait locus (QTL) mapping by pooled-segregant whole genome sequencing has been proved to be a powerful and reliable approach to identify the loci, genes and single nucleotide polymorphism (SNP) variants linked to quantitative traits of yeast.ResultsOne superior thermotolerant industrial strain and one inferior thermosensitive natural strain with distinct high-temperature fermentation performances were screened from 124 Saccharomyces cerevisiae strains as parent strains for crossing and segregant isolation. Based on QTL mapping by pooled-segregant whole genome sequencing as well as the subsequent reciprocal hemizygosity analysis (RHA) and allele replacement analysis, we identified and validated total eight causative genes in four QTLs that linked to high-temperature fermentation of yeast. Interestingly, loss of heterozygosity in five of the eight causative genes including RXT2, ECM24, CSC1, IRA2 and AVO1 exhibited positive effects on high-temperature fermentation. Principal component analysis (PCA) of high-temperature fermentation data from all the RHA and allele replacement strains of those eight genes distinguished three superior parent alleles including VPS34, VID24 and DAP1 to be greatly beneficial to high-temperature fermentation in contrast to their inferior parent alleles. Strikingly, physiological impacts of the superior parent alleles of VPS34, VID24 and DAP1 converged on cell membrane by increasing trehalose accumulation or reducing membrane fluidity.ConclusionsThis work revealed eight novel causative genes and SNP variants closely associated with high-temperature fermentation performance. Among these genes, VPS34 and DAP1 would be good targets for improving high-temperature fermentation of the industrial yeast. It also showed that loss of heterozygosity of causative genes could contribute to the improvement of high-temperature fermentation capacities. Our findings would provide guides to develop more robust and thermotolerant strains for the industrial production of ethanol.

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.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q10 production

In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30°C, pH6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36°C, pH7, and no aeration). By this strategy, the dry cell mass (17.1g/L) and CoQ10 (23.2mg/L) were obtained after 20h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Selection of Thermotolerant Corynebacterium glutamicum Strains for Organic Acid Biosynthesis.

SUMMARYIn recent years, Corynebacterium glutamicum has been considered as producer of many valuable chemical compounds. Among them, organic acids such as l-lactic and succinic acids are the most important ones. It is known that the wild-type C. glutamicum grows well in the temperature range between 25 and 37 °C. Above 40 °C, the biomass growth usually abruptly stops; however, the bacteria remain metabolically active. High temperature affects the metabolic activity of C. glutamicum cells and can lead to changes in the composition and quantity of the fermentation products. Therefore, in a series of subsequent selection steps, we tried to obtain prototrophic strains capable of growing at 44 °C from the culture of homoserine auxotroph C. glutamicum ATCC 13287. During selection, we used complex and mineral media containing succinic and citric acids. As a result, we obtained 47 clones able to grow at elevated temperature. Moreover, the estimated optimal growth temperature for several of them was about 40 °C or higher. Under oxygen limitation conditions, C. glutamicum strains produce organic acids. Regardless of the tested clone, l-lactic acid was the main product. However, its concentration was the highest in the cultures performed at 44 °C. The elevated temperature also affected the biosynthesis of other organic acids. Compared to the parental strain, the concentration of acetic acid increased, and of succinic acid decreased in the cultures of thermotolerant strains. Strain RCG44.3 exhibited interesting properties; it was able to synthesise 27.1 g/L l-lactic acid, with production yield of 0.57 g/g, during 24 h of fermentation at 44 °C.

Biomass and lipid production of a novel freshwater thermo-tolerant mutant strain of Chlorella pyrenoidosa NCIM 2738 in seawater salinity recycled medium

Micro-algae have significant potential for the production of biofuel. It has been previously shown that existing processes of producing micro-algal biofuels are not yet economically viable at commercial scale. Fundamentally, this process will require the implementation of inexpensive harvesting methods and productive algal strains that are tolerant to harsh conditions. The use of freshwater in the large-scale cultivation of microalgae is currently unsustainable; hence, the studies which target the use of seawater medium can reduce the demand of freshwater. Chlorella pyrenoidosa has been extensively studied in freshwater. However, information on the cultivation of C. pyrenoidosa for growth productivity in recycled seawater medium, generated after harvesting through autoflocculation is scarce. In this context, the present study investigates the effect of nutrients sufficient seawater medium following reuse of nutrients exhausted seawater medium on the biochemical composition, biomass and lipid productivities of an in-house developed, thermotolerant strain of C. pyrenoidosa under high temperature (45 °C), outdoor culture conditions. The strain C. pyrenoidosa M18 was found to thrive in seawater medium resulting in increased biomass and lipid productivity. Lipid productivity in seawater medium exceeded 51 mg/Ld−1 which was nearly 2-fold greater than freshwater. Furthermore, the strain was able to grow well and autoflocculate at recycled seawater medium giving biomass and lipid productivities, respectively of 338 mgL−1d−1 and 66 mgL−1d−1. The dry cell weight comprised of 28–32% carbohydrate content when cultivated in recycled seawater medium. Additionally, exposure to high salinity led to an enhanced chlorophyll content as well as increased Fv/Fm ratio. The auto-flocculating property of thermotolerant C. pyrenoidosa M18 strain, in addition to the high biomass and lipid productivities in nutrient exhausted recycled seawater medium, make this microalga an excellent candidate for large-scale outdoor cultivation for biodiesel production.

Oenological traits of Lachancea thermotolerans show signs of domestication and allopatric differentiation

The yeast Lachancea thermotolerans (previously Kluyveromyces thermotolerans) is a species of large, yet underexplored, oenological potential. This study delivers comprehensive oenological phenomes of 94 L. thermotolerans strains obtained from diverse ecological niches worldwide, classified in nine genetic groups based on their pre-determined microsatellite genotypes. The strains and the genetic groups were compared for their alcoholic fermentation performance, production of primary and secondary metabolites and pH modulation in Chardonnay grape juice fermentations. The common oenological features of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and the formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds, used for the first time in a population-scale phenotyping of a non-Saccharomyces yeast, revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, our results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. Together, these observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.

Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

Overexpression of the Bacillus licheniformis GroES enhances thermotolerance of Bacillus subtilis WB600

Use of thermotolerant strains offers the advantage of conducting production process at elevated temperatures, thereby improving the catalytic efficiency and reducing the energy consumption and the production costs. In the present study, thermotolerance gene groES from the thermophilic strain Bacillus licheniformis B186 was implanted into B. subtilis WB600 to improve its thermotolerance. The engineered strain WB600-pHY-groES displayed reduced doubling time as well as increased specific growth rates and cell viability compared with the control. Our results suggested that introducing heat shock proteins (HSPs) from thermophiles into B. subtilis could be an effective approach to enhance its thermotolerance, thus facilitating the development of multichaperone expression systems for constructing more thermotolerant commercial B. subtilis strains. To the best of our knowledge, this is the first report on improving the thermotolerance of B. subtilis by overexpression of HSPs.