RESUMO
Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.
Assuntos
Bacillus subtilis , Consórcios Microbianos , Bacillus subtilis/química , Lipopeptídeos/química , Antifúngicos/químicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) preferentially infects and causes Kaposi's sarcoma (KS) in male patients. However, the biological mechanisms are largely unknown. This study was novel in confirming the extensive nuclear distribution of the androgen receptor (AR) and its co-localization with viral oncoprotein of latency-associated nuclear antigen in KS lesions, indicating a transcription way of AR in KS pathogenesis. The endogenous AR was also remarkably higher in KSHV-positive B cells than in KSHV-negative cells and responded to the ligand treatment of 5α-dihydrotestosterone (DHT), the agonist of AR. Then, the anti-AR antibody-based chromatin immunoprecipitation (ChIP)-associated sequencing was used to identify the target viral genes of AR, revealing that the AR bound to multiple regions of lytic genes in the KSHV genome. The highest peak was enriched in the core promoter sequence of polyadenylated nuclear RNA (PAN), and the physical interaction was verified by ChIP-polymerase chain reaction (PCR) and the electrophoretic mobility shift assay (EMSA). Consistently, male steroid treatment significantly transactivated the promoter activity of PAN in luciferase reporter assay, consequently leading to extensive lytic gene expression and KSHV production as determined by real-time quantitative PCR, and the deletion of nuclear localization signals of AR resulted in the loss of nuclear transport and transcriptional activity in the presence of androgen and thus impaired the expression of PAN RNA. Oncogenically, this study identified that the AR was a functional prerequisite for cell invasion, especially under the context of KSHV reactivation, through hijacking the PAN as a critical effector. Taken together, a novel mechanism from male sex steroids to viral noncoding RNA was identified, which might provide a clue to understanding the male propensity in KS.
Assuntos
RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Receptores Androgênicos/metabolismo , Sarcoma de Kaposi/metabolismo , Caracteres Sexuais , Carcinogênese/metabolismo , Feminino , Herpesvirus Humano 8 , Humanos , Masculino , RNA não Traduzido/metabolismoRESUMO
Saccharomyces cerevisiae is an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion of YPL062W in S. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function of YPL062W and the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered that YPL062W deletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed that YPL062W functions as a core promoter for ALD6 and that the expression level of ALD6 is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062W increases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function of YPL062W but also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCE Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W, demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.
Assuntos
Deleção de Genes , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Metabolismo Secundário/genética , Terpenos/metabolismo , Acetilcoenzima A/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Fermentação , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Ácido Mevalônico/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA , Biologia de SistemasRESUMO
BACKGROUND: Strains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes. RESULTS: Haploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae. CONCLUSIONS: SCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.
Assuntos
Álcalis/metabolismo , Cromossomos Artificiais de Levedura/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Fenótipo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Biologia SintéticaRESUMO
OBJECTIVES: A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. RESULTS: By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18 g/l), and the molar conversion rate was 80.7% within 36 h in 5 l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. CONCLUSIONS: We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.
Assuntos
Ácido Ascórbico/metabolismo , Bacillus megaterium/metabolismo , Fermentação , Gluconobacter oxydans/metabolismo , Consórcios Microbianos , Rhodobacteraceae/metabolismo , Açúcares Ácidos/metabolismo , Bacillus megaterium/crescimento & desenvolvimento , Comunicação Celular , Gluconobacter oxydans/crescimento & desenvolvimento , Interações Microbianas , Rhodobacteraceae/crescimento & desenvolvimento , Vitaminas/metabolismoRESUMO
Microbial consortia, with the merits of strong stability, robustness, and multi-function, played critical roles in human health, bioenergy, and food manufacture, etc. On the basis of 'build a consortium to understand it', a novel microbial consortium consisted of Gluconobacter oxydans, Ketogulonicigenium vulgare and Bacillus endophyticus was reconstructed to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. With this synthetic consortium, 73.7 g/L 2-KGA was obtained within 30 h, which is comparable to the conventional industrial method. A combined time-series proteomic and metabolomic analysis of the fermentation process was conducted to further investigate the cell-cell interaction. The results suggested that the existence of B. endophyticus and G. oxydans together promoted the growth of K. vulgare by supplying additional nutrients, and promoted the 2-KGA production by supplying more substrate. Meanwhile, the growth of B. endophyticus and G. oxydans was compromised from the competition of the nutrients by K. vulgare, enabling the efficient production of 2-KGA. This study provides valuable guidance for further study of synthetic microbial consortia.
Assuntos
Ácido Ascórbico/metabolismo , Metabolômica , Consórcios Microbianos , Proteômica , Açúcares Ácidos/metabolismo , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Fermentação , Gluconobacter oxydans/metabolismo , Microbiologia Industrial , Rhodobacteraceae/metabolismoRESUMO
Defect in the amino acid biosynthetic pathways of Ketogulonicigenium vulgare, the producing strain for 2-keto-L-gulonic acid (2-KGA), is the key reason for its poor growth and low productivity. In this study, five different strains were firstly reconstructed by expressing absent genes in threonine, proline and histidine biosynthetic pathways for better 2-KGA productivity. When mono-cultured in the shake flasks, the strain SyBE_Kv02080002 expressing hsk from Gluconobacter oxydans in threonine biosynthetic pathway achieved the highest biomass and the titer increased by 25.13%. When co-cultured with Bacillus endophyticus, the fermentation cycle decreased by 28.57% than that of the original consortium in 5-L fermenter. Furthermore, reconstruction of threonine biosynthetic pathway resulted in up-regulation of genes encoding sorbosone dehydrogenase and idonate-dehydrogenase, which increased the 2-KGA productivity in SyBE_Kv02080002. This study shows that reconstruction of absent biosynthetic pathways in bacteria is an effective way to enhance the productivity of target products.
Assuntos
Aminoácidos/metabolismo , Bacillus/metabolismo , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Rhodobacteraceae/metabolismo , Açúcares Ácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Meios de Cultura/química , Fermentação , Gluconobacter oxydans/genética , Gluconobacter oxydans/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Sorbose/análogos & derivados , Sorbose/metabolismo , Regulação para CimaRESUMO
Biosynthesis of alkanes in microbial foundries offers a sustainable and green supplement to traditional fossil fuels. The dynamic equilibrium of fatty aldehydes, key intermediates, played a critical role in microbial alkanes production, due to the poor catalytic capability of aldehyde deformylating oxygenase (ADO). In our study, exploration of competitive pathway together with multi-modular optimization was utilized to improve fatty aldehydes balance and consequently enhance alkanes formation in Escherichia coli. Endogenous fatty alcohol formation was supposed to be competitive with alkane production, since both of the two routes consumed the same intermediate-fatty aldehyde. Nevertheless, in our case, alkanes production in E. coli was enhanced from trace amount to 58.8mg/L by the facilitation of moderate fatty alcohol biosynthesis, which was validated by deletion of endogenous aldehyde reductase (AHR), overexpression of fatty alcohol oxidase (FAO) and consequent transcriptional assay of aar, ado and adhP genes. Moreover, alkanes production was further improved to 81.8mg/L, 86.6mg/L or 101.7mg/L by manipulation of fatty acid biosynthesis, lipids degradation or electron transfer system modules, which directly referenced to fatty aldehydes dynamic pools. A titer of 1.31g/L alkanes was achieved in 2.5L fed-batch fermentation, which was the highest reported titer in E. coli. Our research has offered a reference for chemical overproduction in microbial cell factories facilitated by exploring competitive pathway.
Assuntos
Alcanos/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Melhoramento Genético/métodos , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Alcanos/isolamento & purificação , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica/genéticaRESUMO
BACKGROUND: In the industry, the conventional two-step fermentation method was used to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C, by three strains, namely, Gluconobacter oxydans, Bacillus spp. and Ketogulonicigenium vulgare. Despite its high production efficiency, the long incubation period and an additional second sterilization process inhibit the further development. Therefore, we aimed to reorganize a synthetic consortium of G. oxydans and K. vulgare for one-step fermentation of 2-KGA and enhance the symbiotic interaction between microorganisms to perform better. RESULTS: During the fermentation, competition for sorbose of G. oxydans arose when co-cultured with K. vulgare. In this study, the competition between the two microbes was alleviated and their mutualism was enhanced by deleting genes involved in sorbose metabolism of G. oxydans. In the engineered synthetic consortium (H6 + Kv), the yield of 2-KGA (mol/mol) against D-sorbitol reached 89.7 % within 36 h, increased by 29.6 %. Furthermore, metabolomic analysis was used to verify the enhancement of the symbiotic relationship and to provide us potential strategies for improving the synthetic consortium. Additionally, a significant redistribution of metabolism occurred by co-culturing the K. vulgare with the engineered G. oxydans, mainly reflected in the increased TCA cycle, purine, and fatty acid metabolism. CONCLUSIONS: We reorganized and optimized a synthetic consortium of G. oxydans and K. vulgare to produce 2-KGA directly from D-sorbitol. The yield of 2-KGA was comparable to that of the conventional two-step fermentation. The metabolic interaction between the strains was further investigated by metabolomics, which verified the enhancement of the mutualism between the microbes and gave us a better understanding of the synthetic consortium.
Assuntos
Ácido Ascórbico/metabolismo , Fermentação/fisiologia , Consórcios Microbianos/fisiologiaRESUMO
Improving the yield of 2-keto-L-gulonic acid (2-KGA), the direct precursor of vitamin C, draws more and more attention in industrial production. In this study, we try to increase the 2-KGA productivity by computer-aided selection of genes encoding L-sorbose dehydrogenases (SDH) of Ketogulonicigenium vulgare. First, six SDHs were modeled by docking strategy to predict the binding mode with co-factor PQQ. The binding energy between SSDA1-H/SSDA1-L and PQQ was the highest, followed by SSDA3/SSDA2. The binding energy between SSDA1-P/SSDB and PQQ was the lowest. Then, these genes were overexpressed, respectively, in an industrial strain K. vulgare HKv604. Overexpression of ssda1-l and ssda1-h enhanced the 2-KGA production by 7.89 and 12.56 % in mono-cultured K. vulgare, and by 13.21 and 16.86 % when K. vulgare was co-cultured with Bacillus endophyticus. When the engineered K. vulgare SyBE_Kv000116013 (overexpression of ssda1-p) or SyBE_Kv000116016 (overexpression of ssdb) was co-cultured with B. endophyticus, the 2-KGA production decreased significantly. The docking results were in accordance with the experimental data, which indicated that computer-aided modeling is an efficient strategy for screening more efficient enzymes.
Assuntos
Bacillus/fisiologia , Desidrogenases de Carboidrato/química , Rhodobacteraceae/enzimologia , Açúcares Ácidos/metabolismo , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Técnicas de Cocultura , Fermentação , Engenharia Metabólica , Consórcios Microbianos , Simulação de Acoplamento Molecular , Cofator PQQ/química , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismoRESUMO
Engineered microbes offer the opportunity to design and implement artificial molecular pathways for renewable production of tailored chemical commodities. Targeted biosynthesis of odd-chain fatty alcohols is very challenging in microbe, due to the specificity of fatty acids synthase for two-carbon unit elongation. Here, we developed a novel strategy to directly tailor carbon number in fatty aldehydes formation step by incorporating α-dioxygenase (αDOX) from Oryza sativa (rice) into Escherichia coli αDOX oxidizes Cn fatty acids (even-chain) to form Cn-1 fatty aldehydes (odd-chain). Through combining αDOX with fatty acyl-acyl carrier protein (-ACP) thioesterase (TE) and aldehyde reductase (AHR), the medium odd-chain fatty alcohols profile (C11, C13, C15) was firstly established in E. coli. Also, medium even-chain alkanes (C12, C14) were obtained by substitution of AHR to aldehyde decarbonylase (AD). The titer of odd-chain fatty alcohols was improved from 7.4mg/L to 101.5mg/L in tube cultivation by means of fine-tuning endogenous fatty acyl-ACP TE (TesA'), αDOX, AHRs and the genes involved in fatty acids metabolism pathway. Through high cell density fed-batch fermentation, a titer of 1.95g/L odd-chain fatty alcohols was achieved, which was the highest reported titer in E. coli. Our system has greatly expanded the current microbial fatty alcohols profile that provides a new brand solution for producing complex and desired molecules in microbes.
Assuntos
Escherichia coli , Álcoois Graxos/metabolismo , Oryza/genética , Proteínas de Plantas , Escherichia coli/genética , Escherichia coli/metabolismo , Oryza/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genéticaRESUMO
Synthetic biology is an emerging research field that focuses on using rational engineering strategies to program biological systems, conferring on them new functions and behaviours. By developing genetic parts and devices based on transcriptional, translational, post-translational modules, many genetic circuits and metabolic pathways had been programmed in single cells. Extending engineering capabilities from single-cell behaviours to multicellular microbial consortia represents a new frontier of synthetic biology. Herein, we first reviewed binary interaction modes of microorganisms in microbial consortia and their underlying molecular mechanisms, which lay the foundation of programming cell-cell interactions in synthetic microbial consortia. Systems biology studies on cellular systems enable systematic understanding of diverse physiological processes of cells and their interactions, which in turn offer insights into the optimal design of synthetic consortia. Based on such fundamental understanding, a comprehensive array of synthetic microbial consortia constructed in the last decade were reviewed, including isogenic microbial communities programmed by quorum sensing-based cell-cell communications, sender-receiver microbial communities with one-way communications, and microbial ecosystems wired by two-way (bi-directional) communications. Furthermore, many applications including using synthetic microbial consortia for distributed bio-computations, chemicals and bioenergy production, medicine and human health, and environments were reviewed. Synergistic development of systems and synthetic biology will provide both a thorough understanding of naturally occurring microbial consortia and rational engineering of these complicated consortia for novel applications.
Assuntos
Consórcios Microbianos , Biologia Sintética/métodos , HumanosRESUMO
There has been extensive research on the biological recycling of PET waste to address the issue of plastic waste pollution, with ethylene glycol (EG) being one of the main components recovered from this process. Therefore, finding ways to convert PET monomer EG into high-value products is crucial for effective PET waste recycling. In this study, we successfully engineered Escherichia coli to utilize EG and produce glycolic acid (GA), expecting to facilitate the biological recycling of PET waste. The engineered E. coli, able to utilize 10 g/L EG to produce 1.38 g/L GA within 96 h, was initially constructed. Subsequently, strategies based on overexpression of key enzymes and knock-out of the competing pathways are employed to enhance EG utilization along with GA biosynthesis. An engineered E. coli, characterized by the highest GA production titer and substrate conversion rate, was obtained. The GA titer increased to 5.1 g/L with a yield of 0.75 g/g EG, which is the highest level in the shake flake experiments. Transcriptional level analysis and metabolomic analysis were then conducted, revealing that overexpression of key enzymes and knock-out of the competing pathways improved the metabolic flow in the EG utilization. The improved metabolic flow also leads to accelerated synthesis and metabolism of amino acids.
RESUMO
Microbial degradation of petroleum hydrocarbons plays a vital role in mitigating petroleum contamination and heavy oil extraction. In this study, a Saccharomyces cerevisiae capable of degrading hexadecane has been successfully engineered, achieving a maximum degradation rate of up to 20.42%. However, the degradation ability of this strain decreased under various pressure conditions such as high temperature, high osmotic pressure, and acidity conditions. Therefore, a S. cerevisiae with high tolerance to these conditions has been constructed. And then, we constructed an "anti-stress hydrocarbon-degrading" consortium comprising engineered yeast strain SAH03, which degrades hexadecane, and glutathione synthetic yeast YGSH10, which provides stress resistance. This consortium was able to restore the degradation ability of SAH03 under various pressure conditions, particularly exhibiting a significant increase in degradation rate from 5.04% to 17.04% under high osmotic pressure. This study offers a novel approach for improving microbial degradation of petroleum hydrocarbons.
RESUMO
Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.
Assuntos
Bacillus amyloliquefaciens , Proteínas de Bactérias , Engenharia Metabólica , Monoéster Fosfórico Hidrolases , Plasmídeos , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Bacillus amyloliquefaciens/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Técnicas de Inativação de GenesRESUMO
Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.
RESUMO
High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.
Assuntos
Bacillus amyloliquefaciens , Lipopeptídeos , Bacillus amyloliquefaciens/metabolismo , Bacillus amyloliquefaciens/genética , Lipopeptídeos/metabolismo , Tolerância ao Sal , Água do Mar/microbiologia , Alimentos , Perda e Desperdício de AlimentosRESUMO
Pitching ratio has been reported to impact not only on the primary metabolism, but also the secondary metabolism. Comparative metabolomics was used to explore the metabolic responses of Streptomyces lydicus E9 to pitching ratios (1, 10, and 30%, v/v). We identified more than 120 metabolites involved in glycolysis, tricarboxylic acid cycle, and amino acid and secondary metabolism, of which there are significant differences in the quantified 32 metabolites under different pitching ratios by gas chromatography coupled to time-of-flight mass spectrometry. The intracellular levels of most amino acids (e.g., valine, alanine, and isoleucine) declined with the increases of pitching ratios. Especially, the relative abundances of glutamate and proline were not only decreased with the increases of pitching rations, but also had much low level at stages II and III, which might be related to the significant enhancement in streptolydigin of S. lydicus E9 under 30% high pitching ratio. Moreover, principal component analysis revealed that eight metabolites, including glucopyranoside, maltose, cAMP, glycine, proline, lysine, isoleucine, and valine, were considered as potential biomarkers to distinguish the influences of pitching ratios on streptolydigin production. Further investigations demonstrated that the additions of exogenous glutamate and proline (100 mgL⻹) enhanced significantly the accumulation of streptolydigin, indicating that glutamate was the synthetic precursor of streptolydigin, while proline in S. lydicus E9 was converted into glutamate and consequently improved streptolydigin biosynthesis. Therefore, these findings provide new insights into the amino acid responses of S. lydicus E9 to pitching ratios and provide potential strategies to improve streptolydigin production.
Assuntos
Aminoácidos/metabolismo , Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Streptomyces/química , Streptomyces/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , MetabolomaRESUMO
The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.
Assuntos
Aminoglicosídeos/biossíntese , Ácido Glutâmico/metabolismo , Metabolômica , Prolina/metabolismo , Streptomyces/metabolismo , Aminoácidos/biossíntese , Aminoácidos/metabolismo , Antibacterianos/biossíntese , Carbono/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Ácido Glutâmico/farmacologia , Homeostase/efeitos dos fármacos , Poliaminas/metabolismo , Polímeros/metabolismo , Análise de Componente Principal , Prolina/farmacologia , Streptomyces/efeitos dos fármacosRESUMO
Petroleum hydrocarbons are relatively recalcitrant compounds, and as contaminants, they are one of the most serious environmental problems. n-Alkanes are important constituents of petroleum hydrocarbons. Advances in synthetic biology and metabolic engineering strategies have made n-alkane biodegradation more designable and maneuverable for solving environmental pollution problems. In the microbial degradation of n-alkanes, more and more degradation pathways, related genes, microbes, and alkane hydroxylases have been discovered, which provide a theoretical basis for the further construction of degrading strains and microbial communities. In this review, the current advances in the microbial degradation of n-alkanes under aerobic condition are summarized in four aspects, including the biodegradation pathways and related genes, alkane hydroxylases, engineered microbial chassis, and microbial community. Especially, the microbial communities of "Alkane-degrader and Alkane-degrader" and "Alkane-degrader and Helper" provide new ideas for the degradation of petroleum hydrocarbons. Surfactant producers and nitrogen providers as a "Helper" are discussed in depth. This review will be helpful to further achieve bioremediation of oil-polluted environments rapidly.