Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

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.

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.

Optimization and Characterization of an Ultra-Thermostable, Acidophilic, Cellulase-Free Xylanase from a New Obligate Thermophilic Geobacillus thermoleovorans AKNT10 and its Application in Saccharification of Wheat Bran.

Microbial xylanases are enzymes of great importance due to their wide industrial applications, especially in the degradation of lignocellulosic biomass into fermentable sugars. This study aimed to describe the production optimization and partial characterization of an ultra-thermostable, acidophilic, cellulase-free xylanase from an obligate thermophilic eubacterium Geobacillus thermoleovorans strain-AKNT10 (Ac.No. LT158229) isolated from a hot-spring of Puga Valley located at an altitude of 4419 m in Ladakh, India. The optimization of cultural conditions improved enzyme yield by 10.49-fold under submerged fermentation. The addition of 1% (w/v) xylose induced the enzyme synthesis by ~ 165 and 371% when supplemented in the fermentation medium containing wheat bran (WB) 1 and 3%, respectively. The supplementation of sucrose reduced the xylanase production by ~ 25%. Results of partial characterization exhibited that xylanase was optimally active at pH 6.0 and 100 °C. Enzyme retained > 75%, > 83%, and > 84% of activity at 4 °C for 28 days, 100 °C for 60 min, and pHs 3-8 for 60 min, respectively. An outstanding property of AKNT10-xylanase, was the retention of > 71% residual activity at extreme conditions (121 °C and 15 psi pressure) for 15 min. Enzymatic saccharification showed that enzyme was also capable to liberate maximum reducing sugars within 4-8 h under optimized conditions thus it could be a potential candidate for the bioconversion of lignocellulosic biomass as well as other industrial purposes. To the best of our knowledge, this is the first report on such an ultra-thermo-pressure-tolerant xylanase optimally active at pH 6 and 100 °C from the genus Geobacillus.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from Geobacillus kaustophilus.

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The GkHAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that GkHAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global GkHAL folding. These findings suggest that GkHAL could be a promising candidate for potential biotechnological applications.

Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Similarities of Geobacillus bacteria based on their profiles of antimicrobial susceptibility in milk samples.

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus.

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.

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.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

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.

Isolation and Characterization of Lignocellulose-Degrading Geobacillus thermoleovorans from Yellowstone National Park.

The microbial degradation of lignocellulose in natural ecosystems presents numerous biotechnological opportunities, including biofuel production from agricultural waste and feedstock biomass. To explore the degradation potential of specific thermophiles, we have identified and characterized extremophilic microorganisms isolated from hot springs environments that are capable of biodegrading lignin and cellulose substrates under thermoalkaline conditions, using a combination of culturing, genomics, and metabolomics techniques. Organisms that can use lignin and cellulose as a sole carbon source at 60 to 75°C were isolated from sediment slurry of thermoalkaline hot springs (71 to 81°C and pH 8 to 9) of Yellowstone National Park. Full-length 16S rRNA gene sequencing indicated that these isolates were closely related to Geobacillus thermoleovorans. Interestingly, most of these isolates demonstrated biofilm formation on lignin, a phenotype that is correlated with increased bioconversion. Assessment of metabolite level changes in two Geobacillus isolates from two representative springs were undertaken to characterize the metabolic responses associated with growth on glucose versus lignin carbon source as a function of pH and temperature. Overall, results from this study support that thermoalkaline springs harbor G. thermoleovorans microorganisms with lignocellulosic biomass degradation capabilities and potential downstream biotechnological applications. IMPORTANCE Since lignocellulosic biomass represents a major agro-industrial waste and renewable resource, its potential to replace nonrenewable petroleum-based products for energy production is considerable. Microbial ligninolytic and cellulolytic enzymes are of high interest in biorefineries for the valorization of lignocellulosic biomass, as they can withstand the extreme conditions (e.g., high temperature and high pH) required for processing. Of great interest is the ligninolytic potential of specific Geobacillus thermoleovorans isolates to function at a broad range of pH and temperatures, since lignin is the bottleneck in the bioprocessing of lignocellulose. In this study, results obtained from G. thermoleovorans isolates originating from YNP springs are significant because very few microorganisms from alkaline thermal environments have been discovered to have lignin- and cellulose-biodegrading capabilities, and this work opens new avenues for the biotechnological valorization of lignocellulosic biomass at an industrial scale.

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.

Biochemical characterization and detection of antitumor activity of l-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T.

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for l-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta™2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as ∼36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 °C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 °C and 75% at 55 °C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.

A novel method for transforming Geobacillus kaustophilus with a chromosomal segment of Bacillus subtilis transferred via pLS20-dependent conjugation.

BACKGROUND: Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. RESULTS: In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. CONCLUSIONS: This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.

Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1.

ß-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the ß-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli with a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of ß-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R, and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

Transcriptome and growth efficiency comparisons of recombinant thermophiles that produce thermolabile and thermostable proteins: implications for burden-based selection of thermostable proteins.

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.

Genome Annotation of Poly(lactic acid) Degrading Pseudomonas aeruginosa, Sphingobacterium sp. and Geobacillus sp.

Pseudomonas aeruginosa and Sphingobacterium sp. are well known for their ability to decontaminate many environmental pollutants while Geobacillus sp. have been exploited for their thermostable enzymes. This study reports the annotation of genomes of P. aeruginosa S3, Sphingobacterium S2 and Geobacillus EC-3 that were isolated from compost, based on their ability to degrade poly(lactic acid), PLA. Draft genomes of the strains were assembled from Illumina reads, annotated and viewed with the aim of gaining insight into the genetic elements involved in degradation of PLA. The draft genome of Sphinogobacterium strain S2 (435 contigs) was estimated at 5,604,691 bp and the draft genome of P. aeruginosa strain S3 (303 contigs) was estimated at 6,631,638 bp. The draft genome of the thermophile Geobacillus strain EC-3 (111 contigs) was estimated at 3,397,712 bp. A total of 5385 (60% with annotation), 6437 (80% with annotation) and 3790 (74% with annotation) protein-coding genes were predicted for strains S2, S3 and EC-3, respectively. Catabolic genes for the biodegradation of xenobiotics, aromatic compounds and lactic acid as well as the genes attributable to the establishment and regulation of biofilm were identified in all three draft genomes. Our results reveal essential genetic elements that facilitate PLA metabolism at mesophilic and thermophilic temperatures in these three isolates.