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Acetic acid is a prevalent inhibitor in lignocellulosic hydrolysate, which represses microbial growth and bioproduction. Histone modification and chromatin remodeling have been revealed to be critical for regulating eukaryotic metabolism. However, related studies in chronic acetic acid stress responses remain unclear. Our previous studies revealed that overexpression of the histone H4 methyltransferase Set5p enhanced acetic acid stress tolerance of the budding yeast Saccharomyces cerevisiae. In this study, we examined the role of Set5p in acetic acid stress by analyzing global protein expression. Significant activation of intracellular protein expression under the stress was discovered, and the functions of the differential proteins were mainly involved in chromatin modification, signal transduction, and carbohydrate metabolism. Notably, a substantial increase of Set5p expression was observed in response to acetic acid stress. Functional studies demonstrated that the restriction of the telomere capping protein Rtc3p, as well as Ies3p and Taf14p, which are related to chromatin regulation, was critical for yeast stress response. This study enriches the understanding of the epigenetic regulatory mechanisms underlying yeast stress response mediated by histone-modifying enzymes. The results also benefit the development of robust yeast strains for lignocellulosic bioconversion.
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Ácido Acético , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estresse Fisiológico , Ácido Acético/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismoRESUMO
Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.
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Corynebacterium glutamicum , Engenharia Metabólica , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , Glucose/metabolismo , Xilose/metabolismo , Carotenoides/metabolismo , Carbono/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/biossínteseRESUMO
Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: ⢠Global gene transcription of K. marxianus is changed by succinic acid (SA) ⢠Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA ⢠Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.
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Kluyveromyces , Ácido Succínico , Kluyveromyces/genética , Perfilação da Expressão Gênica , TranscriptomaRESUMO
Microproteins, prevalent across all kingdoms of life, play a crucial role in cell physiology and human health. Although global gene transcription is widely explored and abundantly available, our understanding of microprotein functions using transcriptome data is still limited. To mitigate this problem, we present a database, Mip-mining ( https://weilab.sjtu.edu.cn/mipmining/ ), underpinned by high-quality RNA-sequencing data exclusively aimed at analyzing microprotein functions. The Mip-mining hosts 336 sets of high-quality transcriptome data from 8626 samples and nine representative living organisms, including microorganisms, plants, animals, and humans, in our Mip-mining database. Our database specifically provides a focus on a range of diseases and environmental stress conditions, taking into account chemical, physical, biological, and diseases-related stresses. Comparatively, our platform enables customized analysis by inputting desired data sets with self-determined cutoff values. The practicality of Mip-mining is demonstrated by identifying essential microproteins in different species and revealing the importance of ATP15 in the acetic acid stress tolerance of budding yeast. We believe that Mip-mining will facilitate a greater understanding and application of microproteins in biotechnology. Moreover, it will be beneficial for designing therapeutic strategies under various biological conditions.
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Biotecnologia , Transcriptoma , Animais , Humanos , Análise de Sequência de RNA , MicropeptídeosRESUMO
Cellulases and xylanases are plant cell wall-degrading enzymes (CWDEs) that are critical to sustainable bioproduction based on renewable lignocellulosic biomass to reduce carbon dioxide emission. Currently, these enzymes are mainly produced from filamentous fungi, especially Trichoderma reesei and Penicillium oxalicum. However, an in-depth comparison of these two producers has not been performed. Although both P. oxalicum and T. reesei harbor CWDE systems, they exhibit distinct features regulating the production of these enzymes, mainly through different transcriptional regulatory networks. This review presents the strikingly different modes of genome-wide regulation of cellulase and xylanase biosynthesis in P. oxalicum and T. reesei, including sugar transporters, signal transduction cascades, transcription factors, chromatin remodeling, and three-dimensional organization of chromosomes. In addition, different molecular breeding approaches employed, based on the understanding of the regulatory networks, are summarized. This review highlights the existence of very different regulatory modes leading to the efficient regulation of CWDE production in filamentous fungi, akin to the adage that "every road leads to Rome." An understanding of this divergence may help further improvements in fungal enzyme production through the metabolic engineering and synthetic biology of certain fungal species.
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Oleaginous yeasts utilize renewable resources to produce lipids, which benefits sustainable development, and it is of great interest to screen robust lipid producers. Curvibasidium sp. belongs to nonconventional yeast that are very limitedly studied. Here, two cold-adaptive strains of Curvibasidium sp., namely, Y230 and Y231, isolated from the medicinal lichen Usnea diffracta were investigated for their potential in lipid production. Genome mining of Curvibasidium sp. Y231 was performed, and the special features related to fatty acid biosynthesis were revealed. Glucose, xylose, and glycerol were tested as sole carbon sources for yeast cell growth and lipid production. The total lipid contents of Curvibasidium sp. Y230 and Y231 range from 38.43% to 54.62% of the cell dry cell weight at 20°C, and glucose is the optimal carbon source. These results indicate that the Curvibasidium sp. strains are promising for sustainable lipid production. Our study provides basis for exploration of lichen-derived strains for biotechnological applications, and also benefits utilization of other nonconventional yeasts for sustainable production based on genome-based studies.
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Basidiomycota , Líquens , Leveduras , Lipídeos , Glucose , Carbono , BiocombustíveisRESUMO
Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.
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BACKGROUND: Scenedesmus obliquus belongs to green microalgae and is widely used in aquaculture as feed, which is also explored for lipid production and bioremediation. However, genomic studies of this microalga have been very limited. Cell self-flocculation of microalgal cells can be used as a simple and economic method for harvesting biomass, and it is of great importance to perform genome-scale studies for the self-flocculating S. obliquus strains to promote their biotechnological applications. RESULTS: We employed the Pacific Biosciences sequencing platform for sequencing the genome of the self-flocculating microalga S. obliquus AS-6-11, and used the MECAT software for de novo genome assembly. The estimated genome size of S. obliquus AS-6-11 is 172.3 Mbp with an N50 of 94,410 bp, and 31,964 protein-coding genes were identified. Gene Ontology (GO) and KEGG pathway analyses revealed 65 GO terms and 428 biosynthetic pathways. Comparing to the genome sequences of the well-studied green microalgae Chlamydomonas reinhardtii, Chlorella variabilis, Volvox carteri and Micractinium conductrix, the genome of S. obliquus AS-6-11 encodes more unique proteins, including one gene that encodes D-mannose binding lectin. Genes encoding the glycosylphosphatidylinositol (GPI)-anchored cell wall proteins, and proteins with fasciclin domains that are commonly found in cell wall proteins might be responsible for the self-flocculating phenotype, and were analyzed in detail. Four genes encoding both GPI-anchored cell wall proteins and fasciclin domain proteins are the most interesting targets for further studies. CONCLUSIONS: The genome sequence of the self-flocculating microalgal S. obliquus AS-6-11 was annotated and analyzed. To our best knowledge, this is the first report on the in-depth annotation of the S. obliquus genome, and the results will facilitate functional genomic studies and metabolic engineering of this important microalga. The comparative genomic analysis here also provides new insights into the evolution of green microalgae. Furthermore, identification of the potential genes encoding self-flocculating proteins will benefit studies on the molecular mechanism underlying this phenotype for its better control and biotechnological applications as well.
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Chlorella , Microalgas , Scenedesmus , Biomassa , Glicolatos , Microalgas/genéticaRESUMO
Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-ß-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.
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Celulase/genética , Proteínas Fúngicas/genética , Hypocreales/genética , Biossíntese de Proteínas , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hypocreales/metabolismo , Regiões Promotoras Genéticas , Engenharia de Proteínas , Dedos de ZincoRESUMO
Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.
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Biocombustíveis/provisão & distribuição , Etanol/metabolismo , Estudo de Associação Genômica Ampla , Lignina/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Perfilação da Expressão Gênica , Metabolômica , Proteômica , Saccharomyces cerevisiae/genéticaRESUMO
Due to the unique Entner-Doudoroff pathway, Zymomonas mobilis has been acknowledged as a potential host to be engineered for biorefinery to produce biofuels and biobased chemicals. The self-flocculation of Z. mobilis can make the bacterial cells self-immobilized within bioreactors for high density to improve product productivities, and in the meantime enhance their tolerance to stresses, particularly product inhibition and the toxicity of byproducts released during the pretreatment of lignocellulosic biomass. In this work, we explored mechanism underlying such a phenotype with the self-flocculating strain ZM401 developed from the regular non-flocculating strain ZM4. Cellulase de-flocculation and the restoration of the self-flocculating phenotype for the de-flocculated bacterial cells subjected to culture confirmed the essential role of cellulose biosynthesis in the self-flocculation of ZM401. Furthermore, the deactivation of both Type I and Type IV restriction-modification systems was performed for ZM4 and ZM401 to improve their transformation efficiencies. Comparative genome analysis detected the deletion of a thymine from ZMO1082 in ZM401, leading to a frame-shift mutation for the putative gene to be integrated into the neighboring downstream gene ZMO1083 encoding the catalytic subunit A of cellulose synthase, and consequently created a new gene to encode a larger transmembrane protein BcsA_401 for more efficient synthesis of cellulose as well as the development of cellulose fibrils and their entanglement for the self-flocculation of the mutant. These speculations were confirmed by the morphological observation of the bacterial cells under scanning electron microscopy, the impact of the gene deletion on the self-flocculation of ZM401, and the restoration of the self-flocculating phenotype of ZM401 ΔbcsA by the gene complementation. The progress will lay a foundation not only for fundamental research in deciphering molecular mechanisms underlying the self-flocculation of Z. mobilis and stress tolerance associated with the morphological change but also for technological innovations in engineering non-flocculating Z. mobilis and other bacterial species with the self-flocculating phenotype.
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Aderência Bacteriana , Células Imobilizadas/enzimologia , Células Imobilizadas/metabolismo , Celulose/metabolismo , Glucosiltransferases/metabolismo , Zymomonas/enzimologia , Zymomonas/metabolismo , Células Imobilizadas/fisiologia , Enzimas de Restrição-Modificação do DNA , Floculação , Deleção de Genes , Genômica , Glucosiltransferases/genética , Lignina/metabolismo , Engenharia Metabólica , Microscopia Eletrônica de Varredura , Transformação Bacteriana , Zymomonas/citologia , Zymomonas/genéticaRESUMO
BACKGROUND: Saccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve production efficiency under industrially relevant conditions, coordinated expression of multiple genes by manipulating promoter strengths is an efficient approach. It is known that gene expression is highly dependent on the practically used environmental conditions and is subject to dynamic changes. Therefore, investigating promoter activities of S. cerevisiae under different culture conditions in different time points, especially under stressful conditions is of great importance. RESULTS: In this study, the activities of various promoters in S. cerevisiae under stressful conditions and in the presence of xylose were characterized using yeast enhanced green fluorescent protein (yEGFP) as a reporter. The stresses include toxic levels of acetic acid and furfural, and high temperature, which are related to fermentation of lignocellulosic hydrolysates. In addition to investigating eight native promoters, the synthetic hybrid promoter P3xC-TEF1 was also evaluated. The results revealed that P TDH3 and the synthetic promoter P3xC-TEF1 showed the highest strengths under almost all the conditions. Importantly, these two promoters also exhibited high stabilities throughout the cultivation. However, the strengths of P ADH1 and P PGK1 , which are generally regarded as 'constitutive' promoters, decreased significantly under certain conditions, suggesting that cautions should be taken to use such constitutive promoters to drive gene expression under stressful conditions. Interestingly, P HSP12 and P HSP26 were able to response to both high temperature and acetic acid stress. Moreover, P HSP12 also led to moderate yEGFP expression when xylose was used as the sole carbon source, indicating that this promoter could be used for inducing proper gene expression for xylose utilization. CONCLUSION: The results here revealed dynamic changes of promoter activities in S. cerevisiae throughout batch fermentation in the presence of inhibitors as well as using xylose. These results provide insights in selection of promoters to construct S. cerevisiae strains for efficient bioproduction under practical conditions. Our results also encouraged applications of synthetic promoters with high stability for yeast strain development.
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Regulação Fúngica da Expressão Gênica , Microbiologia Industrial , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ácido Acético/farmacologia , Biocombustíveis , Fermentação , Furaldeído/farmacologia , Proteínas de Fluorescência Verde/genética , Temperatura Alta , Saccharomyces cerevisiae/crescimento & desenvolvimento , Estresse Fisiológico , Xilose/químicaRESUMO
Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.
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Proteínas de Bactérias/genética , Proteínas Inativadoras do Complemento/biossíntese , Genoma Bacteriano , Família Multigênica , Sesquiterpenos/metabolismo , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas Inativadoras do Complemento/química , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Alinhamento de Sequência , Sesquiterpenos/química , Streptomyces/química , Streptomyces/classificação , Streptomyces/metabolismoRESUMO
Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.
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Etanol/metabolismo , Glicosídeo Hidrolases/genética , Helianthus/química , Microbiologia Industrial/métodos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Inulina/metabolismo , Microrganismos Geneticamente Modificados/genética , Tubérculos/química , TemperaturaRESUMO
A novel streptomycete, designated as strain DUT 180(T), was isolated from a marine sediment sample collected from a sea cucumber farm in Dalian, northeast China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DUT 180(T) is phylogenetically affiliated to the genus Streptomyces where it formed a distinct phyletic line with recognized Streptomyces species. Morphological and chemotaxonomic data also supported the affiliation of this isolate to the genus Streptomyces. Strain DUT 180(T) was found to exhibit highest sequence similarities of 99.52 and 99.36 % to Streptomyces halophytocola KLBMP 1284(T) and Streptomyces sulphureus NRRL B-1627(T), respectively. However, strain DUT 180(T) could be distinguished from these two closest neighbours by a range of phenotypic properties. The DNA-DNA hybridization analyses between strain DUT 180(T) and the type strains of the phylogenetic neighbours revealed 54.8 ± 1.4 and 52.4 ± 2.8 % relatedness. Based on the phenotypic, chemotaxonomic and phylogenetic evidence, we suggest that the isolate DUT 180(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces xiaopingdaonensis sp. nov. is proposed, with the type strain DUT 180(T) (= KCTC 29679(T) = CGMCC 4.7208(T)).
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Sedimentos Geológicos/microbiologia , Streptomyces/classificação , Streptomyces/isolamento & purificação , Técnicas de Tipagem Bacteriana , China , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/fisiologiaRESUMO
Lignocellulose is an alternative to fossil resources, but its biochemical conversion is not economically competitive. While decentralized processing can reduce logistical cost for this feedstock, sugar platforms need to be developed with energy-saving pretreatment technologies and cost-effective cellulases, and products must be selected correctly. Anaerobic fermentation with less energy consumption and lower contamination risk is preferred, particularly for producing biofuels. Great effort has been devoted to producing cellulosic ethanol, but CO2 released with large quantities during ethanol fermentation must be utilized in situ for credit. Unless titer and yield are improved substantially, butanol cannot be produced as an advanced biofuel. Microbial lipids produced through aerobic fermentation with low yield and intensive energy consumption are not affordable as feedstocks for biodiesel production.
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Etanol , Lignina , Lignina/metabolismo , Etanol/metabolismo , Fermentação , Butanóis , BiocombustíveisRESUMO
Yeast is widely studied in producing biofuels and biochemicals using renewable biomass. Among various yeasts, Saccharomyces cerevisiae has been particularly recognized as an important yeast cell factory. However, economic bioproduction using S. cerevisiae is challenged by harsh environments during fermentation, among which inhibitory chemicals in the culture media or toxic products are common experiences. Understanding the stress-responsive mechanisms is conducive to developing robust yeast strains. Here, we review recent progress in mechanisms underlying yeast stress response, including regulation of cell wall integrity, membrane transport, antioxidative system, and gene transcription. We highlight epigenetic regulation of stress response and summarize manipulation of yeast stress tolerance for improved bioproduction. Prospects in the application of machine learning to improve production efficiency are also discussed.
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Epigênese Genética , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Fermentação , BiocombustíveisRESUMO
Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1, renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1-overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol.
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The Greatwall-family protein kinase Rim15 is associated with the nutrient starvation response, whereas its role in oxidative stress responses remains unclear. Here, acetic acid and peroxide were used as two oxidative stress elicitors. The antioxidant indicator assay under acetic acid stress revealed the impaired growth in rim15Δ related to the regulation of antioxidant systems. Comparative transcriptome analysis revealed that differentially expressed genes (DEGs) are predicted to be mostly regulated by oxidative stress-responsive transcriptional factor Yap1. Among the DEGs, acetic acid stress-induced genes were found, and YAP1 disruption also inhibited their induction. The deletion of Rim15 or the Rim15 kinase domain in yap1Δ did not further decrease the gene expression, suggesting that Rim15 functions together with Yap1 in regulating acetic acid stress-induced genes, which requires Rim15 kinase activity. Additionally, Rim15 regulated H2O2 stress tolerance through partially similar but special mechanisms in that Rim15 kinase activity impacted acetic acid and H2O2 stress tolerance in different degrees, indicating the different mechanisms underlying Rim15-mediated redox regulation against different stressors. These results benefit the better understanding of stress signaling pathways related to Rim15. Given that Rim15 and some of its target genes are conserved across eukaryotes, these results also provide a basis for studies of oxidative stress-related processes in other organisms.
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Anaerobic digestion (AD) is an effective and applicable technology for treating organic wastes to recover bioenergy, but it is limited by various drawbacks, such as long start-up time for establishing a stable process, the toxicity of accumulated volatile fatty acids and ammonia nitrogen to methanogens resulting in extremely low biogas productivities, and a large amount of impurities in biogas for upgrading thereafter with high cost. Microbial electrolysis cell (MEC) is a device developed for electrosynthesis from organic wastes by electroactive microorganisms, but MEC alone is not practical for production at large scales. When AD is integrated with MEC, not only can biogas production be enhanced substantially, but also upgrading of the biogas product performed in situ. In this critical review, the state-of-the-art progress in developing AD-MEC systems is commented, and fundamentals underlying methanogenesis and bioelectrochemical reactions, technological innovations with electrode materials and configurations, designs and applications of AD-MEC systems, and strategies for their enhancement, such as driving the MEC device by electricity that is generated by burning the biogas to improve their energy efficiencies, are specifically addressed. Moreover, perspectives and challenges for the scale up of AD-MEC systems are highlighted for in-depth studies in the future to further improve their performance.