RESUMO
To improve ethanol production directly from CO2 in photosynthetic cyanobacterial systems, one key issue that needs to be addressed is the low ethanol tolerance of cyanobacterial cells. Our previous proteomic and transcriptomic analyses found that several regulatory proteins were up-regulated by exogenous ethanol in Synechocystis sp. PCC6803. In this study, through tolerance analysis of the gene disruption mutants of the up-regulated regulatory genes, we uncovered that one transcriptional regulator, Sll0794, was related directly to ethanol tolerance in Synechocystis. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible regulatory network of Sll0794. The proteomic analysis showed that in the Δsll0794 mutant grown under ethanol stress a total of 54 and 87 unique proteins were down- and up-regulated, respectively. In addition, electrophoretic mobility shift assays demonstrated that the Sll0794 transcriptional regulator was able to bind directly to the upstream regions of sll1514, slr1512, and slr1838, which encode a 16.6 kDa small heat shock protein, a putative sodium-dependent bicarbonate transporter and a carbon dioxide concentrating mechanism protein CcmK, respectively. The study provided a proteomic description of the putative ethanol-tolerance network regulated by the sll0794 gene, and revealed new insights on the ethanol-tolerance regulatory mechanism in Synechocystis. As the first regulatory protein discovered related to ethanol tolerance, the gene may serve as a valuable target for transcription machinery engineering to further improve ethanol tolerance in Synechocystis. All MS data have been deposited in the ProteomeXchange with identifier PXD001266 (http://proteomecentral.proteomexchange.org/dataset/PXD001266).
Assuntos
Proteínas de Bactérias/genética , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Proteoma/genética , Synechocystis/efeitos dos fármacos , Adaptação Fisiológica , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocombustíveis , Etanol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fotossíntese/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , Proteoma/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismo , Transcrição GênicaRESUMO
The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more "reactive" pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.
Assuntos
Oxirredutases do Álcool , Corynebacterium glutamicum , Dissulfetos , Estabilidade Enzimática , Simulação de Dinâmica Molecular , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Dissulfetos/química , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Mutação , Domínio Catalítico , Cinética , Conformação Proteica , Engenharia de Proteínas/métodosRESUMO
The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.
RESUMO
As a special category of pesticides, chiral pesticides have increased the difficulty in investigating pesticide toxicity. Based on their usage, chiral pesticides can be divided into insecticides, herbicides, and fungicides. Over the past decades, great efforts have been made on elucidating their toxicological effects. However, no literature has reviewed the enantioselective toxicity of chiral pesticides since 2014. In recent years, more chiral pesticides have been registered for application. As such, huge research progresses have been achieved in enantioselective toxicity of chiral pesticides. Generally, more researches have remedied the knowledge gap in toxicological effects of old and new chiral pesticides. And the toxicological endpoints being evaluated have become more specific rather than centering on basic toxicity and target organisms. Besides, the underlying mechanisms accounting for the enantioselectivity in toxicological effects of chiral pesticides have been discussed as well. All in all, this review provides the critical knowledge for risk assessments, and help to drive the green-technology of single- or enriched-enantiomer pesticides and formulation of relevant laws and regulations.
Assuntos
Fungicidas Industriais , Herbicidas , Inseticidas , Praguicidas , Praguicidas/toxicidade , Estereoisomerismo , Fungicidas Industriais/toxicidadeRESUMO
Tris (2-ethylhexyl) phosphate (TEHP) is a common organophosphorus flame retardant analog with considerable ecological toxicity. Here, novel strain Ochrobactrum tritici WX3-8 capable of degrading TEHP as the sole C source was isolated. Our results show that the strain's TEHP degradation efficiency reached 75% after 104 h under optimal conditions, i.e., 30 °C, pH 7, bacterial inoculum 3%, and
RESUMO
Knowledge about the substrate scope for a given enzyme is informative for elucidating biochemical pathways and also for expanding applications of the enzyme. However, no general methods are available to accurately predict the substrate specificity of an enzyme. Pyrrolysyl-tRNA synthetase (PylRS) is a powerful tool for incorporating various noncanonical amino acids (NCAAs) into proteins, which enabled us to probe, image, rationally engineer, and evolve protein structure and function. However, the incorporation of a new NCAA typically requires the selection of large libraries of PylRS with randomized mutations at active sites, and this process requires multiple rounds of selection for each new substrate. Therefore, a single aminoacyl-tRNA synthetase with broad substrate promiscuity is ideal to facilitate widespread applications of the genetic NCAA incorporation technique. Herein, machine learning models were developed to predict the substrate specificity of PylRS to accept novel NCAAs that could be incorporated into proteins by three PylRS mutants. The models were built from a training set of 285 unique enzyme-substrate pairs of three PylRS mutants including IFRS, BtaRS, and MFRS against 95 NCAAs. The best BaggingTree (BT) model was then used for virtually screening a NCAAs library containing 1474 phenylalanine, tyrosine, tryptophan, and alanine analogues, and 156 NCAAs were predicted to be accepted by at least one of the three PylRS mutants. Then, 27 NCAAs including 24 positive and 3 negative substrates were experimentally tested for their activities, and 20 of the 24 positive substrates showed weak or strong activity and were accepted by at least one PylRS mutant, among which 11 NCAAs were never reported to be incorporated into proteins before. Three negative substrates did not show any activity. Experimental results suggested that the BT model provides a three-class classification accuracy of 0.69 and a binary classification accuracy of 0.86. This study expanded the substrate scope of three PylRS variants and provided a framework for developing machine learning models to predict substrate specificity of other PylRS variants.
Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Especificidade por Substrato , Alanina , Aminoacil-tRNA Sintetases/genética , Aprendizado de MáquinaRESUMO
Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.
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Survival of photosynthetic cyanobacteria is challenged by environmental contaminations like heavy metals. Among them, deciphering the regulatory mechanisms for cadmium (Cd) in cyanobacteria would facilitate the construction of Cd-resistant strains. In this study, the DNA-Affinity-Purified-chromatin immunoprecipitation assay was employed to identify the direct targets of Sll0649, which was a Cd2+-related response regulator identified in our previous work in model cyanobacteria Synechocystis sp. PCC 6803. As a result, the promoter region of slr0946 encoding the arsenate reductase was enriched fourfolds by quantitative real time PCR analysis. Further, deletion of slr0946 led to a sensitive phenotype to Cd2+ stress compared with the wild type (WT) and the sensitive phenotype of Δslr0946 could be rescued by complementation assay via introducing slr0946 back into Δslr0946. Finally, individually overexpression of slr0946 as well as two Cd2+-related genes identified priviously (i.e., sll1598 and slr0798) in WT could significantly improve the tolerance of Synechocystis sp. PCC 6803 to Cd2+. This study provided a better understanding of the tolerance mechanism to Cd2+ in cyanobacteria and also feasible strategies for tolerance modifications to heavy metals in the future.
RESUMO
Photosynthetic cyanobacteria are sensitive to toxicity of metal cadmium (Cd(2+)). Although metabolic responses against Cd(2+) exposure have been described, the related regulatory mechanism is still unclear in cyanobacteria. In this study, we identified in Synechocystis sp. PCC 6803 a response regulator (RR)-encoding gene sll0649, whose mutant was more sensitive to Cd(2+) stress. Further phenotypic analysis revealed that ∆sll0649 becomes more sensitive to Cu(2+), Fe(2+), Mn(2+) and Zn(2+) stress as well. Using a quantitative iTRAQ-LC-MS/MS proteomics approach, we showed that a total of 156 and 151 unique proteins were down- and up-regulated for at least 2-fold in the ∆sll0649 mutant grown under Cd(2+) stress, respectively. In addition, electrophoretic mobility shift assays showed that Sll0649 was able to bind directly to the upstream regions of sll1598 and slr0798, which encode an Mn(2+) transporter MntC and a Zn(2+) transporting P-type ATPases ZiaA, respectively, suggesting that Sll0649 was involved in Cd(2+) tolerance by regulating and maintaining intracellular metal homeostasis. The involvement of sll1598 and slr0798 genes in Cd(2+) tolerance was also verified by comparative mutant analyses. The study provided a proteomic description of the Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis. BIOLOGICAL SIGNIFICANCE: As a major pollutant on earth, Cd(2+) is toxic to both prokaryotic and eukaryotic organisms. It is thus important to obtain a better understanding of cellular response to Cd(2+) and the related regulatory mechanism. In this study, by screening 44 gene knockout mutants of putative RR-encoding genes in Synechocystis for their sensitivity change to Cd(2+) stress, we identified the orphan RR, Slr0649, involved in Cd(2+) tolerance in Synechocystis. The ∆sll0649 mutant was also found to be more sensitive to high-concentration Cu(2+), Fe(2+), Mn(2+) and Zn(2+), when compared with the wild type. Using an iTRAQ-LC-MSMS based quantitative proteomic analysis coupled with EMSAs, we found that, in addition to its positive regulation on genes directly related to Cd(2+) utilization, Sll0649 can also functions as a key positive regulator either directly or indirectly on expression of multiple genes related to transporting and utilization of several other metal ions. The study provided a proteomic description of the Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.
Assuntos
Cádmio/farmacologia , Regulação Bacteriana da Expressão Gênica , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Tolerância a Medicamentos , Genes Bacterianos/fisiologia , Homeostase , Metais/metabolismo , Metais/farmacologia , Fotossíntese/genética , Proteoma/metabolismo , Proteômica , Estresse Fisiológico , Synechocystis/efeitos dos fármacos , Synechocystis/metabolismoRESUMO
We report here the characterization of a novel orphan response regulator Slr1588 directly involved in the synthesis and transport of compatible solutes against salt stress. In the Δslr1588 mutant, salt tolerance was found to be decreased by 2-3-fold. Using a high performance Q-EXACTIVE hybrid quadrupole-Orbitrap mass spectrometer, we found that proteins involved in the synthesis and transport of glucosylglycerol, a key compatible solute, were up-regulated in the Δslr1588 mutant grown in 4.0% NaCl, suggesting that Slr1588 might function as a repressor for glucosylglycerol metabolism. The functional assignment was further confirmed using an electrophoretic mobility shift assay (EMSA) showing that the purified his-tagged Slr1588 could bind in vitro directly to the upstream regions of sll1566 (ggpS) genes required for glucosylglycerol biosynthesis. In addition, quantitative proteomic analysis showed that the biosynthesis of another key compatible solute in Synechocystis, sucrose, was also up-regulated in the Δslr1588 mutant under 4.0% NaCl, and EMSA showed that the purified his-tagged Slr1588 bound in vitro directly to the upstream regions of sll0045 (spsA) gene required for sucrose biosynthesis. Moreover, proteomic analysis showed that 113 and 127 unique proteins were and up- and down-regulated in the Δslr1588 mutant grown under 4.0% NaCl, respectively. Notably, a dozen transporter genes were down-regulated in the Δslr1588 mutant under salt stress. The study revealed a novel salt-tolerant regulatory mechanism mediated by Slr1588, and also provided a proteomic description of the possible Slr1588 regulon in Synechocystis.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucosídeos/metabolismo , Sacarose/metabolismo , Synechocystis/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Fotossíntese , Proteômica , Tolerância ao SalRESUMO
BACKGROUND: Photosynthetic cyanobacteria have been recently proposed as a 'microbial factory' to produce butanol due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources, respectively. However, to improve the productivity, one key issue needed to be addressed is the low tolerance of the photosynthetic hosts to butanol. RESULTS: In this study, we first applied a quantitative transcriptomics approach with a next-generation RNA sequencing technology to identify gene targets relevant to butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803. The results showed that 278 genes were induced by the butanol exposure at all three sampling points through the growth time course. Genes encoding heat-shock proteins, oxidative stress related proteins, transporters and proteins involved in common stress responses, were induced by butanol exposure. We then applied GC-MS based metabolomics analysis to determine the metabolic changes associated with the butanol exposure. The results showed that 46 out of 73 chemically classified metabolites were differentially regulated by butanol treatment. Notably, 3-phosphoglycerate, glycine, serine and urea related to general stress responses were elevated in butanol-treated cells. To validate the potential targets, we constructed gene knockout mutants for three selected gene targets. The comparative phenotypic analysis confirmed that these genes were involved in the butanol tolerance. CONCLUSION: The integrated OMICS analysis provided a comprehensive view of the complicated molecular mechanisms employed by Synechocystis sp. PCC 6803 against butanol stress, and allowed identification of a series of potential gene candidates for tolerance engineering in cyanobacterium Synechocystis sp. PCC 6803.