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 [...].

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.

Novel Microbial Enzymes with Industrial Applications.

Eberhardt et al [...].

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.

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.

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.

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.

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.

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.

Structural determination of Enzyme-Graphene Nanocomposite Sensor Material.

State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance.

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.

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41 kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80 °C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60 °C for 24 h and over 70% remaining activity after incubation at 70 °C for 24 h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of ß-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

Bioconversion of Beet Molasses to Alpha-Galactosidase and Ethanol.

Molasses are sub-products of the sugar industry, rich in sucrose and containing other sugars like raffinose, glucose, and fructose. Alpha-galactosidases (EC. 3.2.1.22) catalyze the hydrolysis of alpha-(1,6) bonds of galactose residues in galacto-oligosaccharides (melibiose, raffinose, and stachyose) and complex galactomannans. Alpha-galactosidases have important applications, mainly in the food industry but also in the pharmaceutical and bioenergy sectors. However, the cost of the enzyme limits the profitability of most of these applications. The use of cheap sub-products, such as molasses, as substrates for production of alpha-galactosidases, reduces the cost of the enzymes and contributes to the circular economy. Alpha-galactosidase is a specially indicated bioproduct since, at the same time, it allows to use the raffinose present in molasses. This work describes the development of a two-step system for the valuation of beet molasses, based on their use as substrate for alpha-galactosidase and bioethanol production by Saccharomyces cerevisiae. Since this yeast secretes high amounts of invertase, to avoid congest the secretory route and to facilitate alpha-galactosidase purification from the culture medium, a mutant in the SUC2 gene (encoding invertase) was constructed. After a statistical optimization of culture conditions, this mutant yielded a very high rate of molasses bioconversion to alpha-galactosidase. In the second step, the SUC2 wild type yeast strain fermented the remaining sucrose to ethanol. A procedure to recycle the yeast biomass, by using it as nitrogen source to supplement molasses, was also developed.

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 [...].

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.

Heat-Loving ß-Galactosidases from Cultured and Uncultured Microorganisms.

ß-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people, and the generation of galacto-oligosaccharides by transgalactosylation reactions. Due to their thermostability, ß-galactosidases from thermophilic microorganisms are very interesting for industrial processes, as high temperatures can increase the initial productivity of the enzyme, provide higher solubility of substrates, and prevent microbial contamination. In the past, it was necessary to cultivate and grow thermophilic microorganisms to discover novel thermozymes, but the development of metagenomic techniques has allowed researchers to access the genomic potential of uncultivated microbes and their enzymes. The present review gives a brief outline of thermophilic ß-galactosidases, with a special focus on those obtained through metagenomics. Additionally, the sequences of ß-galactosidases found in some public metagenomes from hot springs were studied and compared to other known thermostable ß-galactosidases.