Development of a thermophilic host-vector system for the production of recombinant proteins at elevated temperatures.

Geobacillus spp. are moderate thermophiles that can efficiently produce recombinant proteins. Considering the protein production exhibited by these species, we searched for robust promoters in Geobacillus kaustophilus HTA426. Transcriptome data revealed that several genes were highly expressed during the proliferative phase; their promoters were characterized using reporter assays with Venus fluorescent protein (VFP). The results suggested that the cspD promoter (PcspD) directed robust vfp expression at 60°C in G. kaustophilus. Although cspD potentially encodes a cold-shock protein, PcspD functioned at elevated temperatures. The promoter strongly functioned even in Escherichia coli; this prevented the cloning of some genes (e.g., vfp) downstream of it on a plasmid vector via E. coli-based genetic manipulation. Consequently, we generated a mutated PcspD that functioned inefficiently in E. coli and constructed the pGKE124 plasmid using the mutant promoter. The plasmid could carry vfp in E. coli and afford the production of VFP in G. kaustophilus at a yield of 390 mg/L. pGKE124 directed a similar production in other thermophilic species; the highest yield was observed in Geobacillus thermodenitrificans K1041. Several proteins could be produced using a system involving G. thermodenitrificans K1041 and pGKE124. Notably, the extracellular production of xylanase at a yield of 1 g/L was achieved using this system. Although the leaky production of nonsecretory proteins was observed, we developed a simple process to collectively purify recombinant proteins from the intracellular and extracellular fractions. The findings presented there propose an effective host-vector system for the production of recombinant proteins at elevated temperatures. KEY POINTS: • A thermophilic system to produce recombinant proteins was constructed. • The system produced diverse proteins using inexpensive media at elevated temperatures. • The system produced an extracellular protein at a yield of 1 g/L of culture.

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.

Occurrence of different fucoidanase genes in Flavobacterium sp. SW and enzyme characterization.

Fucoidans are hetero-sulfated polysaccharides that are widely distributed in brown algae and have been extensively studied for their various biological activities. The structure-function relationship of fucoidans remains unclear but can be studied using fucoidan-degrading enzymes (fucoidanases). Here, we isolated and identified Flavobacterium sp. SW as a microbial strain that can grow on fucoidan from Cladosiphon okamuranus as the sole carbon source. Genomic analysis of this strain revealed the presence of two genes, swfct and swfcn2, that are homologous to fct114 from Luteolibacter algae H18 and fcnA from Psychromonas sp. SW5A, respectively. The gene products were produced in Escherichia coli and showed significantly different specificities for fucoidan. Swfct catalyzed the degradation of deacetylated fucoidan from C. okamuranus, and Swfcn2 degraded fucoidans from Saccharina sculpera and Macrocystis pyrifera. The general properties of Swfct were examined by measuring the amounts of reducing ends produced by the enzymatic reaction, and the enzyme properties of Swfcn2 were evaluated by carbohydrate-polyacrylamide gel electrophoresis. Our findings indicate that one microbial strain can harbor genes encoding two different types of fucoidanases.

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.

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426.

Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4-9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition.

A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species.

Geobacillus spp. are moderate thermophiles that have great potential for use in diverse applications. For effective utilization of the species, genetic tools have been extensively studied; however, an overexpression vector remains to be developed. Here we constructed a plasmid vector that can shuttle between Escherichia coli and Geobacillus spp., and which contained a maltose-inducible promoter from Geobacillus kaustophilus HTA426. Although the vector (termed pGKE119) was originally designed for basal gene expression, it surprisingly directed robust protein production in G. kaustophilus. Protein production essentially occurred in an auto-inducible manner without maltose; however, some proteins were produced more efficiently in the presence of maltose. Although the productivity was affected by culture conditions, three proteins were successfully produced with abundance ratios of 12-27% (on a total protein basis) and yields of 77-170 mg (per L culture). pGKE119 directed substantial protein production even in Geobacillus subterraneus, Geobacillus thermoglucosidasius, and Geobacillus thermoleovorans. This suggests that pGKE119 can use a range of Geobacillus spp. as hosts and widely expand their genetic toolbox. Because Geobacillus spp. are highly proliferative bacteria that are distinct from organisms used as protein production hosts, pGKE119 may also provide a novel platform for hyperproduction of recombinant proteins.

Identification and characterization of the fucoidanase gene from Luteolibacter algae H18.

Fucoidan is a hetero-sulfated polysaccharide found in brown algae and has received much attention as an ingredient in functional and health foods. The marine bacterial strain Luteolibacter algae H18 degrades fucoidan from Cladosiphon okamuranus. We purified the fucoidanase from a cell-free extract of L. algae H18, used it to decrease the molecular weight of deacetylated-fucoidan, determined the N-terminal amino acid sequence of the enzyme, and identified the gene involved in the degradation of fucoidan, fct114, in a draft genome sequence of strain H18. The gene product was heterologously produced in Escherichia coli and demonstrated to catalyze the degradation of deacetylated-fucoidan into lower molecular weight fragments. The mass of the gene product Fct114 is 112 kDa (1026 amino acid residues). The general properties of the enzyme were investigated by measuring the amount of reducing ends produced from deacetylated-fucoidan during the reaction. The enzyme was inactive toward fucoidans from other brown seaweed species or toward polysaccharides such as alginic acid, carrageenan, hyaluronic acid, and chondroitin sulfate. The amino acid sequence of Fct114 shared less than 25% identity and had no conserved motifs when compared with previously identified fucoidanases from other marine bacterial strains. These data suggest that Fct114 is a novel polysaccharide-degrading enzyme.

Microbial and genomic characterization of Geobacillus thermodenitrificans OS27, a marine thermophile that degrades diverse raw seaweeds.

Seaweeds are a nonlignocellulosic biomass, but they are often abundant in unique polysaccharides that common microbes can hardly utilize; therefore, polysaccharide degradation is key for the full utilization of seaweed biomass. Here, we isolated 13 thermophiles from seaweed homogenates that had been incubated at high temperature. All of the isolates were Gram-positive and preferentially grew at 60-70 °C. Most formed endospores and were tolerant to seawater salinity. Despite different sources, all isolates were identical regarding 16S rRNA gene sequences and were categorized as Geobacillus thermodenitrificans. Their growth occurred on seaweed polysaccharides with different profiles but required amino acids and/or vitamins, implying that they existed as proliferative cells by utilizing nutrients on seaweed viscous surfaces. Among 13 isolates, strain OS27 was further characterized to show that it can utilize a diverse range of seaweed polysaccharides and hemicelluloses. Notably, strain OS27 degraded raw seaweeds while releasing soluble saccharides. The degradation seemed to depend on enzymes that were extracellularly produced in an inducible manner. The strain could be genetically modified to produce heterologous endoglucanase, providing a transformant that degrades more diverse seaweeds with higher efficiency. The draft sequences of the OS27 genome contained 3766 coding sequences, which included intact genes for 28 glycoside hydrolases and many hypothetical proteins unusual among G. thermodenitrificans. These results suggest that G. thermodenitrificans OS27 serves as a genetic resource for thermostable enzymes to degrade seaweeds and potentially as a microbial platform for high temperature seaweed biorefinery via genetic modification.

Characterization of a novel endo-type alginate lyase derived from Shewanella sp. YH1.

Alginate, which is an anionic polysaccharide, is widely distributed in the cell wall of brown algae. Alginate and the products of its degradation (oligosaccharides) are used in stabilizers, thickeners and gelling agents, especially in the food industry. The degradation of alginate generally involves a combination of several alginate lyases (exo-type, endo-type and oligoalginate lyase). Enhancing the efficiency of the production of alginate degradation products may require the identification of novel alginate lyases with unique characteristics. In this study, we isolated an alginate-utilizing bacterium, Shewanella sp. YH1, from seawater collected off the coast of Tottori prefecture, Japan. The detected novel alginate lyase was named AlgSI-PL7, and was classified in polysaccharide lyase family 7. The enzyme was purified from Shewanella sp. YH1 and a recombinant AlgSI-PL7 was produced in Escherichia coli. The optimal temperature and pH for enzyme activity were around 45°C and 8, respectively. Interestingly, we observed that AlgSI-PL7 was not thermotolerant, but could refold to its active form following an almost complete denaturation at approximately 60°C. Moreover, the degradation of alginate by AlgSI-PL7 produced two to five oligosaccharides, implying this enzyme was an endo-type lyase. Our findings suggest that AlgSI-PL7 may be useful as an industrial enzyme.

Antibiotic resistance mutations induced in growing cells of Bacillus-related thermophiles.

Stress-induced mutagenesis can assist pathogens in generating drug-resistant cells during antibiotic therapy; however, if and how antibiotics induce mutagenesis in microbes remains poorly understood. A non-pathogenic thermophile, Geobacillus kaustophilus HTA426, efficiently produces derivative cells resistant to rifampicin and streptomycin via rpoB and rpsL mutations, respectively. Here, we examined this phenomenon to suggest a novel mutagenic mode induced by antibiotics. Fluctuation analysis indicated that mutations occurred via spontaneous mutations during culture. However, mutations were much more frequent in growing cells than stationary cells, and mutation sites were varied through cell growth. These observations suggested that growing cells induced mutagenesis in response to antibiotics. An in-frame deletion of mfd, which governs transcription-coupled repair to correct DNA lesions on the transcribed strand, caused mutations that were comparable between growing and stationary cells; therefore, the mutagenic mechanism was attributable to DNA repair defects where growing cells depressed mfd function. Mutations occurred more frequently at optimal growth temperatures for G. kaustophilus than at a higher growth temperature, suggesting that the mutagenesis relies on active cellular activities rather than high temperature-associated DNA damage. In addition, the mutagenesis may involve a mutagenic factor targeting these sites, in addition to mfd depression, because rpoB and rpsL mutations were dominant at thymine and guanine sites on the transcribed strand. A similar mutagenic profile was observed for other Geobacillus and thermophilic Bacillus species. This suggests that Bacillus-related thermophiles commonly induce mutagenesis in response to rifampicin and streptomycin to produce resistant cells.

Characterization of a Long-Lived Alginate Lyase Derived from Shewanella Species YH1.

Polysaccharides from seaweeds are widely used in various fields, including the food, biomedical material, cosmetic, and biofuel industries. Alginate, which is a major polysaccharide in brown algae, and the products of its degradation (oligosaccharides) have been used in stabilizers, thickeners, and gelling agents, especially in the food industry. Discovering novel alginate lyases with unique characteristics for the efficient production of oligosaccharides may be relevant for the food and pharmaceutical fields. In this study, we identified a unique alginate lyase derived from an alginate-utilizing bacterium, Shewanella species YH1. The recombinant enzyme (rAlgSV1-PL7) was produced in an Escherichia coli system and it was classified in the Polysaccharide Lyase family 7. The optimal temperature and pH for rAlgSV1-PL7 activity were around 45 °C and 8, respectively. Interestingly, we observed that rAlgSV1-PL7 retained over 80% of its enzyme activity after incubation at 30 °C for at least 20 days, indicating that rAlgSV1-PL7 is a long-lived enzyme. Moreover, the degradation of alginate by rAlgSV1-PL7 produced one to four sugars because of the broad substrate specificity of this enzyme. Our findings suggest that rAlgSV1-PL7 may represent a new commercially useful enzyme.

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity.

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate-FMN-enzyme complex remains unknown. Herein, we report the crystal structures of the DBT-FMN-TdsC and DBTO-FMN-TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Gene identification and characterization of fucoidan deacetylase for potential application to fucoidan degradation and diversification.

Fucoidan is an α-l-fucopyranosyl polymer found in seaweeds with forms that have acetyl and sulfuric modifications and derivatives that are lower and/or diversified, with modifications that have attracted interest as potential bioactive substances. We identified the gene for a fucoidan deacetylase that cleaves acetyl moieties from fucoidan and thereby contributes to fucoidan utilization in the marine bacterium Luteolibacter algae H18. Fucoidan deacetylase was purified to homogeneity from a cell-free extract of L. algae H18, and used to determine the internal amino acid sequence and identify the gene, fud, in a draft genome sequence of the H18 strain. The gene product was heterologously produced in Escherichia coli and was demonstrated to catalyze fucoidan deacetylation, but not desulfation, and degradation into lower forms. In addition to fucoidan deacetylation, the enzyme catalyzed the hydrolysis of p-nitrophenyl esters with organic acids, and p-nitrophenyl acetate was the best substrate among those tested. The present study provides a new tool for fucoidan degradation, potentially expanding investigations on fucoidan derivatives.

Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B.

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer-of-dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9-α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171-1177. © 2017 Wiley Periodicals, Inc.

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae.

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.

Conjugative plasmid transfer from Escherichia coli is a versatile approach for genetic transformation of thermophilic Bacillus and Geobacillus species.

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli-Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10(-7) and 3.8 × 10(-2)/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.

A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αß)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αß)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αß)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αß)-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αß)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.

Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1.

UNLABELLED: The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The '4S' metabolic pathway catalyzes the sequential conversion of DBT to 2-hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D-1 is involved in the first two steps of the '4S' pathway. Here, we determined the first crystal structure of FMN-bound DszC, and found that two distinct conformations occur in the loop region (residues 131-142) adjacent to the active site. On the basis of the DszC-FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed. DATABASE: The atomic coordinates and structure factors for apo-DszC (PDB code: 3X0X) and DszC-FMN (PDB code: 3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).