Potential applications of extremophilic bacteria in the bioremediation of extreme environments contaminated with heavy metals.

Protecting the environment from harmful pollutants has become increasingly difficult in recent decades. The presence of heavy metal (HM) pollution poses a serious environmental hazard that requires intricate attention on a worldwide scale. Even at low concentrations, HMs have the potential to induce deleterious health effects in both humans and other living organisms. Therefore, various strategies have been proposed to address this issue, with extremophiles being a promising solution. Bacteria that exhibit resistance to metals are preferred for applications involving metal removal due to their capacity for rapid multiplication and growth. Extremophiles are a special group of microorganisms that are capable of surviving under extreme conditions such as extreme temperatures, pH levels, and high salt concentrations where other organisms cannot. Due to their unique enzymes and adaptive capabilities, extremophiles are well suited as catalysts for environmental biotechnology applications, including the bioremediation of HMs through various strategies. The mechanisms of resistance to HMs by extremophilic bacteria encompass: (i) metal exclusion by permeability barrier; (ii) extracellular metal sequestration by protein/chelator binding; (iii) intracellular sequestration of the metal by protein/chelator binding; (iv) enzymatic detoxification of a metal to a less toxic form; (v) active transport of HMs; (vi) passive tolerance; (vii) reduced metal sensitivity of cellular targets to metal ions; and (viii) morphological change of cells. This review provides comprehensive information on extremophilic bacteria and their potential roles for bioremediation, particularly in environments contaminated with HMs, which pose a threat due to their stability and persistence. Genetic engineering of extremophilic bacteria in stressed environments could help in the bioremediation of contaminated sites. Due to their unique characteristics, these organisms and their enzymes are expected to bridge the gap between biological and chemical industrial processes. However, the structure and biochemical properties of extremophilic bacteria, along with any possible long-term effects of their applications, need to be investigated further.

[Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review].

The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.

CRISPR/Cas genome editing systems in thermophiles: Current status, associated challenges, and future perspectives.

Thermophiles, offering an attractive and unique platform for a broad range of applications in biofuels and environment protections, have received a significant attention and growing interest from academy and industry. However, the exploration and exploitation of thermophilic organisms have been hampered by the lack of a powerful genome manipulation tool to improve production efficiency. At current, the clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated (Cas) system has been successfully exploited as a competent, simplistic, and powerful tool for genome engineering both in eukaryotes and prokaryotes. Indeed, with the significant efforts made in recent years, some thermostable Cas9 proteins have been well identified and characterized and further, some thermostable Cas9-based editing tools have been successfully established in some representative obligate thermophiles. In this regard, we reviewed the current status and its progress in CRISPR/Cas-based genome editing system towards a variety of thermophilic organisms. Despite the potentials of these progresses, multiple factors/barriers still have to be overcome and optimized for improving its editing efficiency in thermophiles. Some insights into the roles of thermostable CRISPR/Cas technologies for the metabolic engineering of thermophiles as a thermophilic microbial cell factory were also fully analyzed and discussed.

Light-driven bio-decolorization of triphenylmethane dyes by a Clostridium thermocellum-CdS biohybrid.

Widespread application of synthetic dyes could generate colored wastewaters causing a range of serious environmental problems. Due to the complex nature of effluents from textile industries, it is difficult to obtain satisfactory treatment of dyes-contaminated wastewater using one single method. Biohybrids coupling of photocatalysts and biocatalysts have great potential in environmental purification. However, how to select suitable organisms and enhance the hybrid's catalytic activities remain challenging. Here, a novel biohybrid system (Clostridium thermocellum-CdS), created for light-driven biodecolorization under thermophilic treatment by using non-photosynthetic microorganism C. thermocellum self-photosensitized with CdS nanoparticles was established. The biohybrids exhibited remarkable decolorization effects on triphenylmethane dyes. The highest decolorization rate was 0.206 min-1. More importantly, enhanced catalytic activities of cadmium sulfide (CdS)-based biohybrids by controlling the particle sizes of semiconductors were demonstrated. Biohybrids systems (Clostridium thermocellum-CdS) through the self-precipitation of CdS with different particle sizes not only showed dramatic changes in the optical properties but also exhibited a very different decolorization rate. This work can not only further broaden targeted applications of CdS-based biohybrids but also demonstrate a promising route for improving biohybrids corresponding photocatalytic capabilities through in situ precipitation CdS with different particle sizes.

Genome Editing of the Anaerobic Thermophile Thermoanaerobacter ethanolicus Using Thermostable Cas9.

Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism.IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.

Transcriptome analysis of the digestive system of a wood-feeding termite (Coptotermes formosanus) revealed a unique mechanism for effective biomass degradation.

BACKGROUND: Wood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive. RESULTS: In order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus, were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment. CONCLUSION: These findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of future biorefinery.

Impact of orientation and flexibility of peptide linkers on T. maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner.

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)4] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS)4) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80 °C after 5 h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.

Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

Orlistat response to missense mutations in lipoprotein lipase.

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure.

Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores.

Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from Clostridium thermocellum was expressed, identified and displayed on the surface of Bacillus subtilis spores by using the spore coat protein G of B. subtilis as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40 °C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.

Purification and characterization of a hemocyanin (Hemo1) with potential lignin-modification activities from the wood-feeding termite, Coptotermes formosanus Shiraki.

Coptotermes formosanus Shiraki is a well-known wood-feeding termite, which can degrade not only cellulose and hemicellulose polysaccharides, but also some aromatic lignin polymers with its enzyme complex to the woody biomass. In this study, a very abundant protein was discovered and purified, using a three-step column chromatography procedure, from the tissue homogenate of the salivary glands and the gut of C. formosanus. Mass spectrometric analysis and the following peptide searching against the mRNA database toward this termite species indicated that the novel protein was a hemocyanin enzyme, termed as Hemo1, which further exhibited a strong oxidase activity in the substrate bioassays toward ABTS [2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], as well as other aromatic analogues, such as catechol and veratryl alcohols. This oxidative protein was an acid-favored enzyme with a molecular weight at 82 kDa, and highly active at 80 °C. These findings indicated that the novel protein, hemocyanin, discovered from the gut system of C. formosanus, might be an important ligninolytic enzyme involved in the biomass pretreatment processing, which will potentially enhance the digestibility and utilization of biomass polysaccharides in termite digestive systems.

High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein.

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS-PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0.

[Advances in and challenges for thermophilic fermentation of cellulosic ethanol].

Thermophiles can produce cellulosic ethanol at a high temperature where ethanol is directly distillated from fermentation, and biodegradation of lignocellulose can be simultaneously achieved when these thermophiles carry and express cellulase and hemicellulase genes. The simultaneous biodegradation, fermentation and distillation, a three-in-one process, can result in low production costs of cellulosic ethanol. We reviewed the advances and challenges in the approach to the three-in-one process, which refer to lignocellulases, regulation mechanisms, and genetic transfer systems.

Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli.

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ(32)). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ(32). One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ(32). The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.

Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes.

Ethanologenic Thermoanaerobacter species produce ethanol from lignocellulose derived substrates at temperatures above 70 degrees C. In the final steps of ethanol formation, two bifunctional acetaldehyde/alcohol dehydrogenases, AdhB and AdhE, and an alcohol dehydrogenase, AdhA, catalyze redox reactions between acetyl-CoA and ethanol via an acetaldehyde intermediate. DNA cloning and analysis revealed that the dehydrogenase genes and their transcriptional regulatory regions were highly conserved in these species. As determined by real-time PCR, the transcription of adhE was activated by ethanol, while adhB was transcribed without ethanol; however, all of their transcription was reduced at higher ethanol concentrations. Under imitating physiological conditions, AdhE played a crucial role in ethanol formation, and AdhB favored ethanol consumption when ethanol concentration was high e.g. 1%. Thus, the ethanol titer of fermentation is controlled via transcriptional regulation and the properties of specific enzymes in Thermoanaerobacter. These results provide evidence for an ethanol balance model and offer the possibility to raise the ethanol titer by metabolic engineering.