Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis.

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

Using the sulfide-oxidizing bacterium Geobacillus thermodenitrificans to restrict H2S release during chicken manure composting.

Hydrogen sulfide (H2S) is a toxic gas massively released during chicken manure composting. Diminishing its release requires efficient and low cost methods. In recent years, heterotrophic bacteria capable of rapid H2S oxidation have been discovered but their applications in environmental improvement are rarely reported. Herein, we investigated H2S oxidation activity of a heterotrophic thermophilic bacterium Geobacillus thermodenitrificans DSM465, which contains a H2S oxidation pathway composed by sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). This strain rapidly oxidized H2S to sulfane sulfur and thiosulfate. The oxidation rate reached 5.73 µmol min-1·g-1 of cell dry weight. We used G. thermodenitrificans DSM465 to restrict H2S release during chicken manure composting. The H2S emission during composting process reduced by 27.5% and sulfate content in the final compost increased by 34.4%. In addition, this strain prolonged the high temperature phase by 7 days. Thus, using G. thermodenitrificans DSM465 to control H2S release was an efficient and economic method. This study provided a new strategy for making waste composting environmental friendly and shed light on perspective applications of heterotrophic H2S oxidation bacteria in environmental improvements.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Sequence identification and in silico characterization of novel thermophilic lipases from Geobacillus species.

Microbial lipases are utilized in various biotechnological areas, including pharmaceuticals, food, biodiesel, and detergents. In this study, we cloned and sequenced Lip21 and Lip33 genes from Geobacillus sp. GS21 and Geobacillus sp. GS33, then we in silico and experimentally analyzed the encoded lipases. For this purpose, Lip21 and Lip33 were cloned, sequenced, and their amino acid sequences were investigated for determination of biophysicochemical characteristics, evolutionary relationships, and sequence similarities. 3D models were built and computationally affirmed by various bioinformatics tools, and enzyme-ligand interactions were investigated by docking analysis using six ligands. Biophysicochemical property of Lip21 and Lip33 was also determined experimentally and the results demonstrated that they had similar isoelectric point (pI) (6.21) and Tm (75.5°C) values as Tm was revealed by denatured protein analysis of the circular dichroism spectrum and pI was obtained by isoelectric focusing. Phylogeny analysis indicated that Lip21 and Lip33 were the closest to lipases from Geobacillus sp. SBS-4S and Geobacillus thermoleovorans, respectively. Alignment analysis demonstrated that S144-D348-H389 was catalytic triad residues in Lip21 and Lip33, and enzymes possessed a conserved Gly-X-Ser-X-Gly motif containing catalytic serine. 3D structure analysis indicated that Lip21 and Lip33 highly resembled each other and they were α/ß hydrolase-fold enzymes with large lid domains. BANΔIT analysis results showed that Lip21 and Lip33 had higher thermal stability, compared to other thermostable Geobacillus lipases. Docking results revealed that Lip21- and Lip33-docked complexes possessed common residues (H112, K115, Q162, E163, and S141) that interacted with the substrates, except paranitrophenyl (pNP)-C10 and pNP-C12, indicating that these residues might have a significant action on medium and short-chain fatty acid esters. Thus, Lip21 and Lip33 can be potential candidates for different industrial applications.

Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights.

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1.

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable ß-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg-1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.

Insights into the rapid metabolism of Geobacillus sp. LC300: unraveling metabolic requirements and optimal growth conditions.

This study investigated the metabolism of Geobacillus sp. LC300, a promising biorefinery host organism with high substrate utilization rates. A new defined medium was designed and tested that allows for exponential growth to elevated cell densities suitable for quantitative physiological studies. Screening of the metabolic requirements of G. sp. LC300 revealed prototrophy for all essential amino acids and most vitamins and only showed auxotrophy for vitamin B12 and biotin. The effect of temperature and pH on growth rate was investigated, adjusting the optimal growth temperature to several degrees lower than previously reported. Lastly, studies on carbon source utilization revealed a capability for fast growth on several common carbon sources, including monosaccharides, oligosaccharides, and polysaccharides, and the highest ever reported growth rate in defined medium on glucose (2.20 h-1) or glycerol (1.95 h-1). These findings provide a foundation for further exploration of G. sp. LC300's physiology and metabolic regulation, and its potential use in bioproduction processes.

Engineered Geobacillus lipolytic enzymes - Attractive polyesterases that degrade polycaprolactones and simultaneously produce esters.

Plastic pollution is one of the biggest environmental problems plaguing the modern world. Polyester-based plastics contribute significantly to this ecological safety concern. In this study, lipolytic biocatalysts GD-95RM and GDEst-lip developed based on lipase/esterase produced by Geobacillus sp. 95 strain were applied for the degradation of polycaprolactone films (Mn 45.000 (PCL45000) and Mn 80.000 (PCL80000)). The degradation efficiency was significantly enhanced by the addition of short chain alcohols. Lipase GD-95RM (1 mg) can depolymerize 264.0 mg and 280.7 mg of PCL45000 and PCL80000, films respectively, in a 24 h period at 30 °C, while the fused enzyme GDEst-lip (1 mg) is capable of degrading 145.5 mg PCL45000 and 134.0 mg of PCL80000 films in 24 h. The addition of ethanol (25 %) improves the degradation efficiency ~2.5 fold in the case of GD-95RM. In the case of GDEst-lip, 50 % methanol was found to be the optimal alcohol solution and the degradation efficiency was increased by ~3.25 times. The addition of alcohols not only increased degradation speeds but also allowed for simultaneous synthesis of industrially valuable 6-hydroxyhexonic acid esters. The suggested system is an attractive approach for removing of plastic waste and supports the principles of bioeconomics.

Geobacillus Bacteriophages from Compost Heaps: Representatives of Three New Genera within Thermophilic Siphoviruses.

We report a detailed characterization of five thermophilic bacteriophages (phages) that were isolated from compost heaps in Vilnius, Lithuania using Geobacillus thermodenitrificans strains as the hosts for phage propagation. The efficiency of plating experiments revealed that phages formed plaques from 45 to 80 °C. Furthermore, most of the phages formed plaques surrounded by halo zones, indicating the presence of phage-encoded bacterial exopolysaccharide (EPS)-degrading depolymerases. Transmission Electron Microscopy (TEM) analysis revealed that all phages were siphoviruses characterized by an isometric head (from ~63 nm to ~67 nm in diameter) and a non-contractile flexible tail (from ~137 nm to ~150 nm in length). The genome sequencing resulted in genomes ranging from 38,161 to 39,016 bp. Comparative genomic and phylogenetic analysis revealed that all the isolated phages had no close relatives to date, and potentially represent three new genera within siphoviruses. The results of this study not only improve our knowledge about poorly explored thermophilic bacteriophages but also give new insights for further investigation of thermophilic and/or thermostable enzymes of bacterial viruses.

Designing a Specific Pretreatment on Corn Starch to Facilitate Enzymatic Rearrangement of Glycosidic Bonds for Efficiently Reducing Starch Digestibility.

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.

Recombinant expression and characterization of a new laccase, bioinformatically identified, from the Antarctic thermophilic bacterium Geobacillus sp. ID17.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Anoxybacillus: an overview of a versatile genus with recent biotechnological applications.

The Bacillaceae family members are considered to be a good source of microbial factories for biotechnological processes. In contrast to Bacillus and Geobacillus, Anoxybacillus, which would be thermophilic and spore-forming group of bacteria, is a relatively new genus firstly proposed in the year of 2000. The development of thermostable microbial enzymes, waste management and bioremediation processes would be a crucial parameter in the industrial sectors. There has been increasing interest in Anoxybacillus strains for biotechnological applications. Therefore, various Anoxybacillus strains isolated from different habitats have been explored and identified for biotechnological and industrial purposes such as enzyme production, bioremediation and biodegradation of toxic compounds. Certain strains have ability to produce exopolysaccharides possessing biological activities including antimicrobial, antioxidant and anticancer. This current review provides past and recent discoveries regarding Anoxybacillus strains and their potential biotechnological applications in enzyme industry, environmental processes and medicine.

Recombinase polymerase amplification using novel thermostable strand-displacing DNA polymerases from Aeribacillus pallidus and Geobacillus zalihae.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.

Bio-efficacy of Geobacillus thermodenitrificans PS41 against larvicidal, fungicidal, and plant growth-promoting activities.

The microbial interactions with plant hosts were known to establish plant growth and beneficial productivity. Some bacteria, fungi, actinomycetes, yeast, and algae have proven as potential effective microbes in agricultural field. In this study, the insecticidal effect of Geobacillus thermodenitrificans PS41 secondary metabolites was tested against third instar larvae of Spodoptera litura, with mortality rate 60.26 ± 1.5% which might influence the agropest management. The test bacterial metabolites were subjected to GC-MS analysis. Totally, 17 different compounds were identified from the ethyl acetate extract metabolites of PS41 strain. The highest peak was obtained with behenic alcohol compound followed by 1-octadecene and penta erythrityl tetrachloride. The Geobacillus thermodenitrificans PS41 secondary metabolites showed potential antifungal activity against plant pathogenic fungi. The highest inhibit was attained against Cladosporium sp., (25 mm) followed by Rhizoctonia solani and Alternaria brassicola (23 mm). However, no toxic effect was exerted upon earthworm (Perionyx excavatus) when treated with PS41 bacterial metabolites. The potential PS41 strain was also found supporting the plant growth. The potential bacterial strain PS41 did not show antagonistic activity against soil bacteria such as Rhizobium sp., Azotobacter sp., Azospirullum brasilense, Pseudomonas fluorescens and Bacillus megaterium. The potential test organism, Geobacillus thermodenitrificans PS41, possessing biopesticide and biofertilizer properties can be a suitable ecofriendly organic applicant in agricultural field for enhancing crop production.

Structural and functional analyses of GH51 alpha-L-arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability.

Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid-base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C-C bond between C171 and C213 by becoming closer to each other.

Monoacylglycerol lipase from marine Geobacillus sp. showing lysophospholipase activity and its application in efficient soybean oil degumming.

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 µm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

Crystal Structure of 4,6-α-Glucanotransferase GtfC-ΔC from Thermophilic Geobacillus 12AMOR1: Starch Transglycosylation in Non-Permuted GH70 Enzymes.

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permutation of the catalytic domain, and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains. Despite these differences, the first crystal structure of a GtfC, GbGtfC-ΔC from Geobacillus 12AMOR1, and the first one representing a non-permuted GH70 enzyme, reveals high structural similarity in the core domains with most GtfBs, featuring a similar tunneled active site. We propose that GtfC (and related GtfD) enzymes evolved from starch-degrading α-amylases from GH13 by acquiring α-1,6 transglycosylation capabilities, before the events that resulted in circular permutation of the catalytic domain observed in other GH70 enzymes (glucansucrases, GtfB-type α-GTs). AlphaFold modeling and sequence alignments suggest that the GbGtfC structure represents the GtfC subfamily, although it has a so far unique alternating α-1,4/α-1,6 product specificity, likely determined by residues near acceptor binding subsites +1/+2.

New Platform for Screening Genetic Libraries at Elevated Temperatures: Biological and Genomic Information and Genetic Tools of Geobacillus thermodenitrificans K1041.

Geobacillus thermodenitrificans K1041 is an unusual thermophile that is highly transformable via electroporation, making it a promising host for screening genetic libraries at elevated temperatures. In this study, we determined its biological properties, draft genome sequence, and effective vectors and also optimized the electroporation procedures in an effort to enhance its utilization. The organism exhibited swarming motility but not detectable endospore formation, and growth was rapid at 60°C under neutral and relatively low-salt conditions. Although the cells showed negligible acceptance of shuttle plasmids from general strains of Escherichia coli, methylation-controlled plasmids from dam mutant strains were efficiently accepted, suggesting circumvention of a restriction-modification system in G. thermodenitrificans K1041. We optimized the electroporation procedure to achieve efficiencies of 103 to 105 CFU/µg for five types of plasmids, which exhibited the different copy numbers and segregational stabilities in G. thermodenitrificans K1041. Some sets of plasmids were compatible. Moreover, we observed substantial plasmid-directed production of heterologous proteins in the intracellular or extracellular environments. Our successful construction of a library of promoter mutants using K1041 cells as hosts and subsequent screening at elevated temperatures to identify improved promoters revealed that G. thermodenitrificans K1041 was practical as a library host. The draft genomic sequence of the organism contained 3,384 coding genes, including resA and mcrB genes, which are involved in restriction-modification systems. Further examination revealed that in-frame deletions of resA increased transformation efficiencies, but mcrB deletion had no effect. The ΔresA mutant exhibited transformation efficiencies of >105 CFU/µg for some plasmids. IMPORTANCE Geobacillus thermodenitrificans K1041 has yet to be fully characterized. Although it is transformable via electroporation, it rarely accepts Escherichia coli-derived plasmids. This study clarified the biological and genomic properties of G. thermodenitrificans K1041. Additionally, we developed an electroporation procedure resulting in efficient acceptance of E. coli-derived plasmids. This procedure produced transformants using small amounts of plasmids immediately after the ligation reaction. Thus, G. thermodenitrificans K1041 was identified as a host for screening promoter mutants at elevated temperatures. Furthermore, because this strain efficiently produced heterologous proteins, it could serve as a host for screening thermostable proteins encoded in random mutant libraries or metagenomes. We also generated a ΔresA mutant that exhibited transformation efficiencies of >105 CFU/µg, which were highest in cases of electroporation-based transformation of Geobacillus spp. with E. coli-derived plasmids. Our findings provide a new platform for screening diverse genetic libraries at elevated temperatures.