RESUMEN
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.
Asunto(s)
Aciltransferasas , Amoníaco-Liasas , Vías Biosintéticas , Coenzima A Ligasas , Escherichia coli , Flavanonas , Flavanonas/biosíntesis , Flavanonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vías Biosintéticas/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Ingeniería Metabólica/métodos , Ácidos Cumáricos/metabolismo , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Tirosina/metabolismoRESUMEN
Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli, and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana, three enzyme cascades FjTAL-SruCAR-YqhD, FjTAL-SruCAR-YqhD-HpaBC, and FjTAL-SruCAR-YqhD-HpaBC-COMT were constructed to produce 1028.5 mg/L p-coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.
Asunto(s)
Escherichia coli , Ingeniería Metabólica , Tirosina , Tirosina/metabolismo , Tirosina/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Flavobacterium/metabolismo , Flavobacterium/enzimología , Flavobacterium/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Lignina/metabolismo , Lignina/química , Amoníaco-Liasas/metabolismo , Amoníaco-Liasas/genética , Amoníaco-Liasas/química , FenolesRESUMEN
BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.
Asunto(s)
Dióxido de Carbono , Cupriavidus necator , Fermentación , Ingeniería Metabólica , Resveratrol , Cupriavidus necator/metabolismo , Cupriavidus necator/genética , Resveratrol/metabolismo , Dióxido de Carbono/metabolismo , Ingeniería Metabólica/métodos , Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Amoníaco-Liasas/metabolismo , Amoníaco-Liasas/genética , Vías BiosintéticasRESUMEN
p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.
Asunto(s)
Amoníaco-Liasas , Flavobacterium , Flavobacterium/genética , Amoníaco-Liasas/genética , Amoníaco-Liasas/química , TirosinaRESUMEN
Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product-which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.
Asunto(s)
Amoníaco-Liasas , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Ácidos Cumáricos/metabolismo , Ingeniería de Proteínas , Tirosina/metabolismoRESUMEN
p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TALclu ) has the highest affinity (Km =0.019â mm) and conversion efficiency (kcat /Km= 1631 s-1 â mm-1 ) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03â g/L after 8â hours by TALclu and 2.35â g/L after 24â h by TAL from Rivularia sp. PCC 7116 (TALrpc ) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.
Asunto(s)
Amoníaco-Liasas , Escherichia coli , Amoníaco-Liasas/genética , Ácidos Cumáricos , Escherichia coli/metabolismo , Fenilanina Amoníaco-Liasa , Filogenia , Tirosina/metabolismoRESUMEN
p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 â) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.
Asunto(s)
Amoníaco-Liasas , Amoníaco-Liasas/genética , Amoníaco-Liasas/química , Ácidos Cumáricos , Escherichia coli/genética , TirosinaRESUMEN
The liver is a crucial center in the regulation of energy homeostasis under starvation. Although downregulation of mammalian target of rapamycin complex 1 (mTORC1) has been reported to play pivotal roles in the starvation responses, the underpinning mechanisms in particular upstream factors that downregulate mTORC1 remain largely unknown. To identify genetic variants that cause liver energy disorders during starvation, we conduct a zebrafish forward genetic screen. We identify a liver hulk (lvh) mutant with normal liver under feeding, but exhibiting liver hypertrophy under fasting. The hepatomegaly in lvh is caused by enlarged hepatocyte size and leads to liver dysfunction as well as limited tolerance to starvation. Positional cloning reveals that lvh phenotypes are caused by mutation in the ftcd gene, which encodes the formimidoyltransferase cyclodeaminase (FTCD). Further studies show that in response to starvation, the phosphorylated ribosomal S6 protein (p-RS6), a downstream effector of mTORC1, becomes downregulated in the wild-type liver, but remains at high level in lvh. Inhibition of mTORC1 by rapamycin rescues the hepatomegaly and liver dysfunction of lvh. Thus, we characterize the roles of FTCD in starvation response, which acts as an important upstream factor to downregulate mTORC1, thus preventing liver hypertrophy and dysfunction.
Asunto(s)
Amoníaco-Liasas/genética , Glutamato Formimidoiltransferasa/genética , Hepatomegalia/genética , Hígado/metabolismo , Enzimas Multifuncionales/genética , Proteína S6 Ribosómica/genética , Animales , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatomegalia/metabolismo , Hepatomegalia/patología , Humanos , Hígado/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Complejos Multiproteicos/genética , Mutación/genética , Fosforilación , Transducción de Señal/genética , Inanición/genética , Inanición/metabolismo , Inanición/patología , Pez Cebra/genéticaRESUMEN
We report here the complete genome sequence of the Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. To assemble a complete genome, we obtained short reads from Illumina sequencing and long reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus, we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL region that has the signature of a new opine catabolic gene cluster including the three genes that encode the three subunits of an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL-DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in the synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes.
Asunto(s)
Cromosomas/genética , Genoma Bacteriano , Plásmidos/genética , Rhizobium/genética , Aminoácidos/genética , Amoníaco-Liasas/genética , Mapeo Cromosómico , Biología Computacional , ADN Bacteriano , Oxazinas , Enfermedades de las Plantas/microbiología , Microbiología del Suelo , Secuenciación Completa del GenomaRESUMEN
p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.
Asunto(s)
Amoníaco-Liasas , Escherichia coli , Amoníaco-Liasas/genética , Ácidos Cumáricos , Escherichia coli/genética , Propionatos , Rhodotorula , Tirosina/genéticaRESUMEN
Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factor was the introduction of multiple pipA genes into the whole-cell system. As a result, we produced 724 mM pipecolic acid (72.4 % conversion), and the productivity was 0.78 g/L/h from 1 M l-lysine after 5 days. This is the highest production reported to date.
Asunto(s)
Amoníaco-Liasas/genética , Escherichia coli/metabolismo , Ácidos Pipecólicos/metabolismo , Amoníaco-Liasas/metabolismo , Biotransformación , Medios de Cultivo/química , Escherichia coli/genética , Fermentación , Expresión Génica , Lisina/análisis , Lisina/metabolismo , Ingeniería Metabólica , Metales/análisis , Metales/metabolismo , Secuencias Repetidas en Tándem , Factores de TiempoRESUMEN
Pyruvate is an important pharmaceutical intermediate and is widely used in food, nutraceuticals, and pharmaceuticals. However, high environmental pollution caused by chemical synthesis or complex separation process of microbial fermentation methods constrain the supply of pyruvate. Here, one-step pyruvate and d-alanine production from d,l-alanine by whole-cell biocatalysis was investigated. First, l-amino acid deaminase (Pm1) from Proteus mirabilis was expressed in Escherichia coli, resulting in pyruvate titer of 12.01 g/L. Then, N-terminal coding sequences were introduced to the 5'-end of the pm1 gene to enhance the expression of Pm1 and the pyruvate titer increased to 15.13 g/L. Next, product utilization by the biocatalyst was prevented by knocking out the pyruvate uptake transporters (cstA, btsT) and the pyruvate metabolic pathway genes pps, poxB, pflB, ldhA, and aceEF using CRISPR/Cas9, yielding 30.88 g/L pyruvate titer. Finally, by optimizing the reaction conditions, the pyruvate titer was further enhanced to 43.50 g/L in 8 H with a 79.99% l-alanine conversion rate; meanwhile, the resolution of d-alanine reached 84.0%. This work developed a whole-cell biocatalyst E. coli strain for high-yield, high-efficiency, and low-pollution pyruvate and d-alanine production, which has great potential for the commercial application in the future.
Asunto(s)
Alanina/metabolismo , Amoníaco-Liasas , Proteínas Bacterianas , Escherichia coli , Microorganismos Modificados Genéticamente , Proteus mirabilis/genética , Ácido Pirúvico/metabolismo , Amoníaco-Liasas/biosíntesis , Amoníaco-Liasas/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Microorganismos Modificados Genéticamente/enzimología , Proteus mirabilis/enzimologíaRESUMEN
BACKGROUND: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction. RESULTS: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved. CONCLUSIONS: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.
Asunto(s)
Técnicas de Cocultivo/métodos , Ácidos Cumáricos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Resveratrol/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Aciltransferasas/genética , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Arabidopsis/enzimología , Basidiomycota/enzimología , Corismato Mutasa/genética , Corismato Mutasa/metabolismo , Codón/genética , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Escherichia coli/crecimiento & desarrollo , Fermentación , Genes Fúngicos , Genes de Plantas , Ingeniería Genética , Microbiología Industrial , Malonil Coenzima A/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Prefenato Deshidrogenasa/genética , Prefenato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Tirosina/metabolismo , Vitis/enzimologíaRESUMEN
Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.
Asunto(s)
Evolución Molecular , Regulación Fúngica de la Expresión Génica , ARN Mensajero/metabolismo , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Perfilación de la Expresión Génica , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , TranscriptomaRESUMEN
p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from deamination of L-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 µM-1 min-1, while Sts-TAL had a much higher efficiency with a kcat/Km value of 78.3 µM-1 min-1. Both Sts-TAL and Sas-TAL can also take L-phenylalanine as the substrate to yield trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)-1, which represents the highest efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.
Asunto(s)
Actinobacteria/enzimología , Amoníaco-Liasas/metabolismo , Ácidos Cumáricos/metabolismo , Secuencia de Aminoácidos , Amoníaco-Liasas/química , Amoníaco-Liasas/genética , Biocatálisis , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Cinética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , Tirosina/metabolismoRESUMEN
In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine-ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure-function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated â¼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase-ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.
Asunto(s)
Amoníaco-Liasas/química , Anabaena/química , Proteínas Bacterianas/química , Hidrolasas/química , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Anabaena/genética , Anabaena/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hidrolasas/genética , Hidrolasas/metabolismo , Modelos Moleculares , Mutación , Conformación Proteica , Multimerización de Proteína , Especificidad por SustratoRESUMEN
As synthetic biology and metabolic engineering tools improve, it is feasible to construct more complex microbial synthesis systems that may be limited by the machinery and resources available in an individual cell. Coculture fermentation is a promising strategy for overcoming these constraints by distributing objectives between subpopulations, but the primary method for controlling the composition of the coculture of production systems has been limited to control of the inoculum composition. We have developed a quorum sensing (QS)-based growth-regulation circuit that provides an additional parameter for regulating the composition of a coculture over the course of the fermentation. Implementation of this tool in a naringenin-producing coculture resulted in a 60% titer increase over a system that was optimized by varying inoculation ratios only. We additionally demonstrated that the growth control circuit can be implemented in combination with a communication module that couples transcription in one subpopulation to the cell-density of the other population for coordination of behavior, resulting in an additional 60% improvement in naringenin titer.
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Técnicas de Cocultivo/métodos , Flavanonas/metabolismo , Ingeniería Metabólica/métodos , Percepción de Quorum/genética , Amoníaco-Liasas/genética , Amoníaco-Liasas/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ligasas/genética , Ligasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Percepción de Quorum/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteína Fluorescente RojaRESUMEN
BACKGROUND: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. RESULTS: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive. CONCLUSIONS: The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.
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Amoníaco-Liasas/metabolismo , Ácidos Cafeicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/metabolismo , Ingeniería Metabólica , Amoníaco-Liasas/genética , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Glucosa/metabolismo , Oxidación-ReducciónRESUMEN
Accumulating statistics have shown that liver cancer causes the second highest mortality rate of cancer-related deaths worldwide, of which 80% is hepatocellular carcinoma (HCC). Given the underlying molecular mechanism of HCC pathology is not fully understood yet, identification of reliable predictive biomarkers is more applicable to improve patients' outcomes. The results of principal component analysis (PCA) showed that the grouped data from 1557 samples in Gene Expression Omnibus (GEO) came from different populations, and the mean tumor purity of tumor tissues was 0.765 through the estimate package in R software. After integrating the differentially expressed genes (DEGs), we finally got 266 genes. Then, the protein-protein interaction (PPI) network was established based on these DEGs, which contained 240 nodes and 1747 edges. FOXM1 was the core gene in module 1 and highly associated with FOXM1 transcription factor network pathway, while FTCD was the core gene in module 2 and was enriched in the metabolism of amino acids and derivatives. The expression levels of hub genes were in line with The Cancer Genome Atlas (TCGA) database. Meanwhile, there were certain correlations among the top ten genes in the up- and downregulated DEGs. Finally, Kaplan-Meier curves and receiver operating characteristic (ROC) curves were plotted for the top five genes in PPI. Apart from CDKN3, the others were closely concerned with overall survival. In this study, we detected the potential biomarkers and their involved biological processes, which would provide a new train of thought for clinical diagnosis and treatment.
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Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transcriptoma/genética , Amoníaco-Liasas/genética , Carcinoma Hepatocelular/patología , Proteínas Cdc20/genética , Biología Computacional , Ciclina A2/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Supervivencia sin Enfermedad , Fosfatasas de Especificidad Dual/genética , Proteína Forkhead Box M1/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutamato Formimidoiltransferasa/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/patología , Enzimas Multifuncionales/genética , Pronóstico , Mapas de Interacción de Proteínas/genética , Programas InformáticosRESUMEN
BACKGROUND: Hypertension and diabetes have been associated with inefficient arsenic metabolism, primarily through studies undertaken in populations exposed through drinking water. Recently, rice has been recognized as a source of arsenic exposure, but it remains unclear whether populations with high rice consumption but no known water exposure are at risk for the health problems associated with inefficient arsenic metabolism. METHODS: The relationships between arsenic metabolism efficiency (% inorganic arsenic, % monomethylarsenate and % dimethylarsinate in urine) and three hypertension- and seven diabetes-related traits were estimated among 12 609 participants of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). A two-sample Mendelian randomization approach incorporated genotype-arsenic metabolism relationships from literature, and genotype-trait relationships from HCHS/SOL, with a mixed-effect linear model. Analyses were stratified by rice consumption and smoking. RESULTS: Among never smokers with high rice consumption, each percentage point increase in was associated with increases of 1.96 mmHg systolic blood pressure (P = 0.034) and 1.85 mmHg inorganic arsenic diastolic blood pressure (P = 0.003). Monomethylarsenate was associated with increased systolic (1.64 mmHg/percentage point increase; P = 0.021) and diastolic (1.33 mmHg/percentage point increase; P = 0.005) blood pressure. Dimethylarsinate, a marker of efficient metabolism, was associated with lower systolic (-0.92 mmHg/percentage point increase; P = 0.025) and diastolic (-0.79 mmHg/percentage point increase; P = 0.004) blood pressure. Among low rice consumers and ever smokers, the results were consistent with no association. Evidence for a relationship with diabetes was equivocal. CONCLUSIONS: Less efficient arsenic metabolism was associated with increased blood pressure among never smokers with high rice consumption, suggesting that arsenic exposure through rice may contribute to high blood pressure in the Hispanic/Latino community.