Characterization, whole-genome sequence analysis, and protease production of a new thermophilic Bacillus licheniformis strain isolated from Debagh hot spring, Algeria.

A new thermophilic strain, designated as Bacillus sp. LMB3902, was isolated from Hammam Debagh, the hottest spring in Algeria (up to 98 °C). This isolate showed high protease production in skim milk media at 55 °C and exhibited significant specific protease activity by using azocasein as a substrate (157.50 U/mg). Through conventional methods, chemotaxonomic characteristics, 16S rRNA gene sequencing, and comparative genomic analysis with the closely related strain Bacillus licheniformis DSM 13 (ATCC 14580 T), the isolate Bacillus sp. LMB3902 was identified as a potentially new strain of Bacillus licheniformis. In addition, the gene functions of Bacillus sp. LMB3902 strain were predicted using the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups, Non-Redundant Protein Sequence Database, Swiss-Prot, and Pfam databases. The results showed that the genome size of Bacillus sp. LMB3902 was 4.279.557 bp, with an average GC content of 46%. The genome contained 4.760 predicted genes, including 8 rRNAs, 78 tRNAs, and 24 sRNAs. A total of 235 protease genes were annotated including 50 proteases with transmembrane helix structures and eight secreted proteases with signal peptides. Additionally, the majority of secondary metabolites found by antiSMASH platform showed low similarity to identified natural products, such as fengicin (53%), lichenysin (57%), and surfactin (34%), suggesting that this strain may encode for novel uncharacterized natural products which can be useful for biotechnological applications. This study is the first report that describes the complete genome sequence, taxono-genomics, and gene annotation as well as protease production of the Bacillus genus in this hydrothermal vent.

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer.

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents. KEY POINTS: • A novel thermostable esterase has been retrieved through functional metagenomics • The esterase is detergent-tolerant, which is attractive for some applications • The esterase can be expressed in a yeast mesophilic host to enhance its yield.

Insights on Microbial Communities Inhabiting Non-Volcanic Hot Springs.

The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resulting in more available niches and broader microbial diversity. Nonetheless, certain metabolic pathways were attributed to less abundant members of the microbial community, reinforcing the idea that the rare biosphere plays important roles in the network of interactions present in an ecosystem and acts as genetic reservoirs. In addition, three of the assembled MAGs represent novel microbial diversity found in this hot spring. Moreover, the presence of enzymes and microorganisms with possible biotechnological applications was confirmed, including proteases, lipases and cell-wall degrading enzymes, pointing to the potential for the hot spring as a source for thermozymes.

Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases.

Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, ß-glucosidases, xylanases, and ß-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein's high temperature optima, broad pH range, and thermostability further highlights its biotechnological potential.

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase.

BACKGROUND: The recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market. RESULTS: The gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium. CONCLUSIONS: A K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

Biobutanol from cheese whey.

At present, due to environmental and economic concerns, it is urgent to evolve efficient, clean and secure systems for the production of advanced biofuels from sustainable cheap sources. Biobutanol has proved better characteristics than the more widely used bioethanol, however the main disadvantage of biobutanol is that it is produced in low yield and titer by ABE (acetone-butanol-ethanol) fermentation, this process being not competitive from the economic point of view. In this review we summarize the natural metabolic pathways for biobutanol production by Clostridia and yeasts, together with the metabolic engineering efforts performed up to date with the aim of either enhancing the yield of the natural producer Clostridia or transferring the butanol production ability to other hosts with better attributes for industrial use and facilities for genetic manipulation. Molasses and starch-based feedstocks are main sources for biobutanol production at industrial scale hitherto. We also review herewith (and for the first time up to our knowledge) the research performed for the use of whey, the subproduct of cheese making, as another sustainable source for biobutanol production. This represents a promising alternative that still needs further research. The use of an abundant waste material like cheese whey, that would otherwise be considered an environmental pollutant, for biobutanol production, makes economy of the process more profitable.

Editorial for Special Issue "Yeasts Biochemistry and Biotechnology".

Yeasts, both Saccharomyces and non-conventional strains, are currently the focus of active research due to their impressive applications in biotechnological bioprocesses, such as being hosts to produce recombinant proteins or main actors of the fermentation industries [...].

Novel Microbial Enzymes with Industrial Applications.

Eberhardt et al [...].

Characterization of a novel thermophilic metagenomic GH5 endoglucanase heterologously expressed in Escherichia coli and Saccharomyces cerevisiae.

BACKGROUND: Endoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases. RESULTS: Sequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by ultrafiltration, with a significantly higher yield of 531.95 U/mL, revealing a much more suitable system for production of large amounts of the enzyme. Their biochemical characterization revealed biotechnologically interesting enzymes. Both Cel776Ec and Cel776Sc had an optimal temperature of 80 °C and optimal pH of 5. Cel776Ec exhibited high thermostability maintaining its activity for 24 h at 60 °C and maintained its activity longer than Cel776Sc at increasing incubation temperatures. Moreover, its substrate specificity allowed the degradation of both cellulose and xylan. Whereas Cel776Ec was more active in the presence of calcium and magnesium, manganese was found to increase Cel776Sc activity. A stronger inhibitory effect was found for Cel776Ec than Cel776Sc adding detergent SDS to the reaction mix, whereas EDTA only significantly affected Cel776Sc activity. CONCLUSIONS: Our study reports the discovery of a new promising biocatalyst for its application in processes, such as the production of biofuel and the saccharification of plant biomass, due to its bifunctional enzymatic activity as an endoglucanase and as a xylanase, as well as highlights the advantages of a yeast expression system over bacteria.

Reactivity of a Recombinant Esterase from Thermus thermophilus HB27 in Aqueous and Organic Media.

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S hydrolysed triglycerides of various acyl chains, which is a rare characteristic among carboxylic ester hydrolases from extreme thermophiles, with maximum activity on tributyrin. It also displayed interfacial activation towards triacetin. KLEST-3S was also tested as a biocatalyst in organic media. The esterase provided high yields for the acetylation of alcohols. In addition, KLEST-3S catalyzed the stereoselective hydrolysis of (R,S)-ibuprofen methyl ester (87% ee). Our results indicate that KLEST-3S may be a robust and efficient biocatalyst for application in industrial bioconversions.

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts.

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable ß-galactosidases.

In the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin-Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-ß-D-galactopyranoside (ONPG) and p-Nitrophenyl-ß-D-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

Comparative Metagenomic Analysis of Two Hot Springs From Ourense (Northwestern Spain) and Others Worldwide.

With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas and Muiño da Veiga are two important human-use hot springs, previously studied with traditional culture methods, but never explored with a metagenomic approach. In the present study, we have performed metagenomic sequence-based analyses to compare the taxonomic composition and functional potential of these hot springs. Proteobacteria, Deinococcus-Thermus, Firmicutes, Nitrospirae, and Aquificae are the dominant phyla in both geothermal springs, but there is a significant difference in the abundance of these phyla between As Burgas and Muiño da Veiga. Phylum Proteobacteria dominates As Burgas ecosystem while Aquificae is the most abundant phylum in Muiño da Veiga. Taxonomic and functional analyses reveal that the variability in water geochemistry might be shaping the differences in the microbial communities inhabiting these geothermal springs. The content in organic compounds of As Burgas water promotes the presence of heterotrophic populations of the genera Acidovorax and Thermus, whereas the sulfate-rich water of Muiño da Veiga favors the co-dominance of genera Sulfurihydrogenibium and Thermodesulfovibrio. Differences in ammonia concentration exert a selective pressure toward the growth of nitrogen-fixing bacteria such as Thermodesulfovibrio in Muiño da Veiga. Temperature and pH are two important factors shaping hot springs microbial communities as was determined by comparative analysis with other thermal springs.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.

Ixr1p regulates oxygen-dependent HEM13 transcription.

In Saccharomyces cerevisiae, HEM13 encodes the enzyme coproporphyrinogen III oxidase, which catalyzes the rate-limiting step in heme biosynthesis. HEM13 is a regulated hypoxic gene repressed by Rox1p and Mot3p under aerobic conditions. In this study, we further investigate the hypoxic expression of HEM13, focusing on the promoter regions that are functionally important during hypoxia and on the effect of deleting the transcriptional regulators Sut1p, Sut2p, Upc2p, Ecm22p and Ixr1p. Ixr1p is necessary for the high expression of HEM13 under hypoxic conditions and its function is exerted in vivo through the HEM13 promoter region extending from -577 to -419. Ixr1p binds in vivo to the HEM13 promoter both under aerobic and under hypoxic conditions. Purified Ixr1p binds in vitro to two sequences extending from -534 to -509 and from -497 to -450, respectively. These DNA regions compete for Ixr1p binding and the consensus KTTSAAYKGTTYASA is important for the regulatory protein to interact. These results suggest that the regulation of HEM13 expression is dependent on two proteins with high mobility group (HMG) domains: Rox1p and Ixr1p. Their interactions with the HEM13 promoter might change in the transition from aerobiosis to hypoxia.

Editorial for the Special Issue: Thermophiles and Thermozymes.

Heat-loving microorganisms or thermophiles arouse noticeable scientific interest nowadays, not only with the aim to elucidate the mystery of life at high temperatures, but also due to the huge field of biotechnological applications of the enzymes they produce or thermozymes, able to function under industrial harsh conditions [...].