RESUMO
So far, a variety of metabolite components of kiwifruit have been elucidated. However, the identification and analysis of flavonoids in different tissues of kiwifruit are rarely carried out. In this study, we performed transcriptome and metabolome analyses of roots (Gkf_R), stems (Gkf_T), leaves (Gkf_L), and fruits (Gkf_F) to provide insights into the differential accumulation and regulation mechanisms of flavonoids in kiwifruit. Results showed that a total of 301 flavonoids were identified, in four tissues with different accumulation trends, and a large proportion of flavonoids had high accumulation in Gkf_L and Gkf_R. A total of 84 genes have been identified involved in the flavonoid biosynthesis pathway, and the expression levels of five LAR, two DFR, and one HCT were significantly correlated with the accumulation of 16 flavonoids and co-localized in the flavonoid biosynthesis pathway. In addition, a total of 2362 transcription factor genes were identified, mainly MYBs, bHLHs, ERFs, bZIPs and WRKYs, among which the expression level of bHLH74, RAP2.3L/4L/10L, MYB1R1, and WRKY33 were significantly correlated with 25, 56, 43, and 24 kinds of flavonoids. Our research will enrich the metabolomic data and provide useful information for the directed genetic improvement and application in the pharmaceutical industry of kiwifruit.
Assuntos
Actinidia , Flavonoides , Regulação da Expressão Gênica de Plantas , Metaboloma , Transcriptoma , Actinidia/genética , Actinidia/metabolismo , Flavonoides/biossíntese , Flavonoides/metabolismo , Frutas/metabolismo , Frutas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica/métodos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Vias Biossintéticas/genética , Metabolômica/métodos , Folhas de Planta/metabolismo , Folhas de Planta/genéticaRESUMO
BACKGROUND: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation. RESULTS: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively. CONCLUSION: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.
Assuntos
Escherichia coli , Fermentação , Glucose , Glicólise , Engenharia Metabólica , Escherichia coli/metabolismo , Escherichia coli/genética , Engenharia Metabólica/métodos , Glucose/metabolismo , Vias Biossintéticas , Regiões Promotoras Genéticas , MalatosRESUMO
Ten new drimane meroterpenoids talarines A-J (1-10), along with six known analogues (11-16), were isolated from desert soil-derived fungus Talaromyces pinophilus LD-7. Their 2D structures were elucidated by comprehensive interpretation of NMR and HRESIMS data. Electronic circular dichroism calculation was used to establish their absolute configurations. Compounds 2, 10, and 11 showed antiviral activities toward vesicular stomatitis virus with IC50 values of 18, 15, and 23 nM, respectively. The structure-bioactivity relationship indicated that chlorine substitution at C-5 contributed greatly to their antiviral activities. Finally, we identified a new halogenase outside the biosynthetic gene cluster, which was responsible for C-5 halogenation of the precursor isocoumarin 17 as a tailoring step in chlorinated meroterpenoids assembly.
Assuntos
Antivirais , Talaromyces , Talaromyces/química , Antivirais/farmacologia , Antivirais/química , Antivirais/isolamento & purificação , Estrutura Molecular , Terpenos/farmacologia , Terpenos/química , Terpenos/isolamento & purificação , Vias Biossintéticas , Relação Estrutura-Atividade , Halogenação , Sesquiterpenos Policíclicos/farmacologiaRESUMO
Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum, we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes - p-coumaroyl CoA 2'-hydroxylase (C2'H), C-prenyltransferase (C-PT), and cyclase - originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants.
Assuntos
Apiaceae , Vias Biossintéticas , Cumarínicos , Filogenia , Cumarínicos/metabolismo , Vias Biossintéticas/genética , Apiaceae/genética , Apiaceae/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Evolução Molecular , Duplicação GênicaRESUMO
Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: ⢠Best enzyme and strain combination were selected for de novo naringenin production. ⢠After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. ⢠This de novo production is the highest reported so far in E. coli.
Assuntos
Aciltransferases , Amônia-Liases , Vias Biossintéticas , Coenzima A Ligases , Escherichia coli , Flavanonas , Flavanonas/biossíntese , Flavanonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vias Biossintéticas/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Amônia-Liases/genética , Amônia-Liases/metabolismo , Engenharia Metabólica/métodos , Ácidos Cumáricos/metabolismo , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Tirosina/metabolismoRESUMO
BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.
Assuntos
Antocianinas , Mirtilos Azuis (Planta) , Saccharomyces cerevisiae , Antocianinas/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mirtilos Azuis (Planta)/genética , Mirtilos Azuis (Planta)/metabolismo , Engenharia Metabólica/métodos , Vias Biossintéticas , Redes e Vias Metabólicas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
KEY MESSAGE: Transcriptomics and phenotypic data analysis identified 24 transcription factors (TFs) that play key roles in regulating the competitive accumulation of lignin and flavonoids. Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts, including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into 3 clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into 2 clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2 and TtWRKY15) were involved in flavonoids and lignin biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between flavonoid and lignin biosynthesis pathway during the development of T. tuan seeds, that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuan seeds.
Assuntos
Flavonoides , Regulação da Expressão Gênica de Plantas , Lignina , Sementes , Lignina/metabolismo , Lignina/biossíntese , Flavonoides/metabolismo , Flavonoides/biossíntese , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica , Transcriptoma/genética , Redes Reguladoras de Genes , Genes de Plantas , Vias Biossintéticas/genéticaRESUMO
Rehmannia glutinosa produces many phenylethanoid glycoside (PhG) compounds, including salidroside, which not only possesses various biological activities but also is a core precursor of some medicinal PhGs, so it is very important to elucidate the species' salidroside biosynthesis pathway to enhance the production of salidroside and its derivations. Although some plant copper-containing amine oxidases (CuAOs), phenylacetaldehyde reductases (PARs) and UDP-glucose glucosyltransferases (UGTs) are thought to be vital catalytic enzymes involved in the downstream salidroside biosynthesis pathways, to date, none of these proteins or the associated genes in R. glutinosa have been characterized. To verify a postulated R. glutinosa salidroside biosynthetic pathway starting from tyrosine, this study identified and characterized a set of R. glutinosa genes encoding RgCuAO, RgPAR and RgUGT enzymes for salidroside biosynthesis. The functional activities of these proteins were tested in vitro by heterologous expression of these genes in Escherichia coli, confirming these catalytic abilities in these corresponding reaction steps of the biosynthetic pathway. Importantly, four enzyme-encoding genes (including the previously reported RgTyDC2 encoding tyrosine decarboxylase and the RgCuAO1, RgPAR1 and RgUGT2 genes) were cointegrated into Saccharomyces cerevisiae to reconstitute the R. glutinosa salidroside biosynthetic pathway, achieving an engineered strain that produced salidroside and validating these enzymes' catalytic functions. This study elucidates the complete R. glutinosa salidroside biosynthesis pathway from tyrosine metabolism in S. cerevisiae, establishing a basic platform for the efficient production of salidroside and its derivatives.
Assuntos
Vias Biossintéticas , Glucosídeos , Fenóis , Rehmannia , Saccharomyces cerevisiae , Fenóis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Rehmannia/genética , Rehmannia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 µg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.
Assuntos
Lactuca , Lolium , Lolium/genética , Lolium/efeitos dos fármacos , Lolium/crescimento & desenvolvimento , Lolium/metabolismo , Lactuca/efeitos dos fármacos , Lactuca/genética , Lactuca/crescimento & desenvolvimento , Família Multigênica , Festuca/genética , Festuca/metabolismo , Festuca/microbiologia , Festuca/efeitos dos fármacos , Festuca/crescimento & desenvolvimento , Fungicidas Industriais/farmacologia , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Vias Biossintéticas , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Estrutura Molecular , Genoma Fúngico , Ascomicetos/genética , Ascomicetos/efeitos dos fármacos , Ascomicetos/metabolismo , Fusarium/efeitos dos fármacos , Fusarium/genética , Fusarium/crescimento & desenvolvimentoRESUMO
Siderophores are small molecule iron chelators. The entomopathogenic fungus Beauveria bassiana produces a plethora of siderophores under iron-limiting conditions. In this study, a siderophore biosynthesis pathway, akin to the general pathway observed in filamentous fungi, was revealed in B. bassiana. Among the siderophore biosynthesis genes (SID), BbSidA was required for the production of most siderophores, and the SidC and SidD biosynthesis gene clusters were indispensable for the production of ferricrocin and fusarinine C, respectively. Biosynthesis genes play various roles in siderophore production, vegetative growth, stress resistance, development, and virulence, in which BbSidA plays the most important role. Accordingly, B. bassiana employs a cocktail of siderophores for iron metabolism, which is essential for fungal physiology and host interactions. This study provides the initial network for the genetic modification of siderophore biosynthesis, which not only aims to improve the efficacy of biocontrol agents but also ensures the efficient production of siderophores.
Assuntos
Beauveria , Vias Biossintéticas , Proteínas Fúngicas , Sideróforos , Beauveria/metabolismo , Beauveria/genética , Sideróforos/metabolismo , Sideróforos/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ferro/metabolismo , Animais , Insetos/microbiologia , Família Multigênica , Ferricromo/análogos & derivadosRESUMO
Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.
Assuntos
Sideróforos , Sideróforos/biossíntese , Sideróforos/química , Sideróforos/metabolismo , Espectrometria de Massas em Tandem/métodos , Desferroxamina/metabolismo , Desferroxamina/química , Cromatografia Líquida/métodos , Vias Biossintéticas , Família Multigênica , Ferro/metabolismo , Ferro/química , Meios de Cultura/química , Meios de Cultura/metabolismoRESUMO
The sequencing of microbial genomes has far outpaced their functional annotation. Stable isotopic labeling can be used to link biosynthetic genes with their natural products; however, the availability of the required isotopically substituted precursors can limit the accessibility of this approach. Here, we describe a method for using inverse stable isotopic labeling (InverSIL) to link biosynthetic genes with their natural products. With InverSIL, a microbe is grown on an isotopically substituted medium to create a fully substituted culture, and subsequently, the incorporation of precursors of natural isotopic abundance can be tracked by mass spectrometry. This eliminates issues with isotopically substituted precursor availability. We demonstrate the utility of this approach by linking a luxI-type acyl-homoserine lactone synthase gene in a bacterium that grows on methanol with its quorum sensing signal products. In the future, InverSIL can also be used to link biosynthetic gene clusters hypothesized to produce siderophores with their natural products.
Assuntos
Produtos Biológicos , Marcação por Isótopo , Marcação por Isótopo/métodos , Produtos Biológicos/metabolismo , Produtos Biológicos/química , Família Multigênica , Percepção de Quorum/genética , Espectrometria de Massas/métodos , Vias Biossintéticas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Isótopos de Carbono/químicaRESUMO
1, 3-propanediol (1, 3-PDO) is an important diol with wide applications in the pharmaceutical, food, and cosmetics industries. In addition, 1, 3-PDO serves as a crucial monomer in the synthesis of polytrimethylene terephthalate, an important synthetic fiber material. Microbial conversion of renewable resources such as glucose into 1, 3-PDO has been industrialized due to its environmentally friendly, energy-efficient, safe, and sustainable characteristics. It serves as a successful case in the design and application of microbial cell factories for biochemicals. However, concerns such as food scarcity and climate change are driving the exploration of non-food, low-cost, and sustainable alternatives as biomanufacturing feedstocks. The biosynthesis of 1, 3-PDO from the C3 feedstock glycerol by microorganisms has been well studied. In recent years, increasing attention has been paid to the synthesis of 1, 3-PDO from C1 feedstocks such as methanol, which has higher energy density than glucose and glycerol. Several new artificial biosynthetic pathways have been proposed and validated, laying a foundation for the sustainable bioproduction of 1, 3-PDO. This article reviews the feedstock transition from C6 to C3 and C1 carbon sources for the microbial synthesis of 1, 3-PDO and discusses the strategies for reprogramming metabolic pathway to enhance 1, 3-PDO biosynthesis from different feedstocks. Finally, the development prospects of 1, 3-PDO bioproduction from C1 feedstocks are forecasted.
Assuntos
Carbono , Propilenoglicóis , Carbono/metabolismo , Propilenoglicóis/metabolismo , Glicerol/metabolismo , Microbiologia Industrial , Glucose/metabolismo , Engenharia Metabólica , Metanol/metabolismo , Vias Biossintéticas , Fermentação , Bactérias/metabolismoRESUMO
Vitamins, as indispensable organic compounds in life activities, demonstrate a complex and refined metabolic network in organisms. This network involves the coordination of multiple enzymes and the integration of various metabolic pathways. Despite the achievements in metabolic engineering and catalytic mechanism research, the lack of studies regarding detailed enzymatic properties for a large number of key enzymes limits the enhancement of vitamin production efficiency and hinders the in-depth understanding and optimization of vitamin synthesis mechanisms. Such limitations not only restrict the industrial application of vitamins but also impede the development of related bio-technologies. This study comprehensively reviews the research progress in the enzymes involved in vitamin biosynthesis and details the current status of research on the enzymes of 13 vitamin synthesis pathways, including their catalytic mechanisms, kinetic properties, and applications in biology. In addition, this study compares the properties of enzymes involved in vitamin metabolic pathways and the glycolysis pathway, and reveals the characteristics of catalytic efficiency and substrate affinity of enzymes in vitamin synthesis pathways. Furthermore, this study discusses the potential and prospects of applying deep learning methods to the research on properties of enzymes associated with vitamin biosynthesis, giving new insights into the production and optimization of vitamins.
Assuntos
Redes e Vias Metabólicas , Vitaminas , Vitaminas/biossíntese , Vitaminas/metabolismo , Vias Biossintéticas , Enzimas/metabolismo , Engenharia Metabólica/métodos , GlicóliseRESUMO
Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD+-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.
Assuntos
Reatores Biológicos , Fermentação , Succinatos , Yarrowia , Yarrowia/metabolismo , Yarrowia/genética , Succinatos/metabolismo , Engenharia Metabólica/métodos , Nitrogênio/metabolismo , Vias Biossintéticas , Isocitrato Desidrogenase/metabolismo , Isocitrato Desidrogenase/genéticaRESUMO
Terbinafine, fluconazole, and amorolfine inhibit fungal ergosterol synthesis by acting on their target enzymes at different steps in the synthetic pathway, causing the accumulation of various intermediates. We found that the effects of these three in- hibitors on yeast morphology were different. The number of morphological parameters commonly altered by these drugs was only approximately 6% of the total. Using a rational strategy to find commonly changed parameters,we focused on hidden essential similarities in the phenotypes possibly due to decreased ergosterol levels. This resulted in higher apparent morphological similarity. Improvements in morphological similarity were observed even when canonical correlation analysis was used to select biologically meaningful morphological parameters related to gene function. In addition to changes in cell morphology, we also observed differences in the synergistic effects among the three inhibitors and in their fungicidal effects against pathogenic fungi possibly due to the accumulation of different intermediates. This study provided a comprehensive understanding of the properties of inhibitors acting in the same biosynthetic pathway.
Assuntos
Antifúngicos , Ergosterol , Fenótipo , Ergosterol/metabolismo , Ergosterol/biossíntese , Antifúngicos/farmacologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Fluconazol/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Terbinafina/farmacologiaRESUMO
As one of the most essential types of heterocyclic compounds, pyrazines have a characteristic smell and taste and have a wide range of commercial applications, especially in the food industry. With the development of the food industry, the demand for pyrazines has increased. Therefore, understanding the properties, functions, and synthetic pathways of pyrazines is one of the fundamental methods to produce, control, and apply pyrazines in food or medical systems. In this review, we provide an overview of the synthesis pathways and physiological or pharmacological functions of naturally occurring pyrazines. In particular, we focus on the biosynthesis and pharmacological effects of 2,3,5,6-Tetramethylpyrazine (TTMP), 2,5-Dimethylpyrazine (2,5-DMP), and 2,3,5-trimethylpyrazine (TMP). Furthermore, areas where further research on pyrazines is needed are discussed in this work.
Assuntos
Pirazinas , Pirazinas/química , Pirazinas/farmacologia , Pirazinas/síntese química , Humanos , Animais , Vias Biossintéticas/efeitos dos fármacosRESUMO
INTRODUCTION: Fungi biosynthesize chemically diverse secondary metabolites with a wide range of biological activities. Natural product scientists have increasingly turned towards bioinformatics approaches, combining metabolomics and genomics to target secondary metabolites and their biosynthetic machinery. We recently applied an integrated metabologenomics workflow to 110 fungi and identified more than 230 high-confidence linkages between metabolites and their biosynthetic pathways. OBJECTIVES: To prioritize the discovery of bioactive natural products and their biosynthetic pathways from these hundreds of high-confidence linkages, we developed a bioactivity-driven metabologenomics workflow combining quantitative chemical information, antiproliferative bioactivity data, and genome sequences. METHODS: The 110 fungi from our metabologenomics study were tested against multiple cancer cell lines to identify which strains produced antiproliferative natural products. Three strains were selected for further study, fractionated using flash chromatography, and subjected to an additional round of bioactivity testing and mass spectral analysis. Data were overlaid using biochemometrics analysis to predict active constituents early in the fractionation process following which their biosynthetic pathways were identified using metabologenomics. RESULTS: We isolated three new-to-nature stemphone analogs, 19-acetylstemphones G (1), B (2) and E (3), that demonstrated antiproliferative activity ranging from 3 to 5 µM against human melanoma (MDA-MB-435) and ovarian cancer (OVACR3) cells. We proposed a rational biosynthetic pathway for these compounds, highlighting the potential of using bioactivity as a filter for the analysis of integrated-Omics datasets. CONCLUSIONS: This work demonstrates how the incorporation of biochemometrics as a third dimension into the metabologenomics workflow can identify bioactive metabolites and link them to their biosynthetic machinery.
Assuntos
Vias Biossintéticas , Fungos , Metabolômica , Família Multigênica , Humanos , Metabolômica/métodos , Fungos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismoRESUMO
Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio-activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains.
Assuntos
Vias Biossintéticas , Deleção de Genes , Metaboloma , Streptomyces coelicolor , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Vias Biossintéticas/genética , Lipidômica , Triglicerídeos/metabolismo , Triglicerídeos/biossínteseRESUMO
Vitamin D deficiencies are linked to multiple human diseases. Optimizing its synthesis, physicochemical properties, and delivery systems while minimizing side effects is of clinical relevance and is of great medical and industrial interest. Biotechnological techniques may render new modified forms of vitamin D that may exhibit improved absorption, stability, or targeted physiological effects. Novel modified vitamin D derivatives hold promise for developing future therapeutic approaches and addressing specific health concerns related to vitamin D deficiency or impaired metabolism, such as avoiding hypercalcemic effects. Identifying and engineering key enzymes and biosynthetic pathways involved, as well as developing efficient cultures, are therefore of outmost importance and subject of intense research. Moreover, we elaborate on the critical role that microbial bioconversions might play in the a la carte design, synthesis, and production of novel, more efficient, and safer forms of vitamin D and its analogs. In summary, the novelty of this work resides in the detailed description of the physiological, medical, biochemical, and epidemiological aspects of vitamin D supplementation and the steps towards the enhanced and simplified industrial production of this family of bioactives relying on microbial enzymes. KEY POINTS: ⢠Liver or kidney pathologies may hamper vitamin D biosynthesis ⢠Actinomycetes are able to carry out 1α- or 25-hydroxylation on vitamin D precursors.