Efficient synthesis of eriodictyol from L-tyrosine in Escherichia coli.
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ABSTRACT:
SUBMITTER: Zhu S
PROVIDER: S-EPMC4018925 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAcetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production.ResultsHere, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain.ConclusionsTyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.
| S-EPMC6423740 | biostudies-literature
Project description:Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/μg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.
| S-EPMC10957790 | biostudies-literature
Project description:Phenylacetic acid (PAA) is a fine chemical with a high industrial demand for its widespread uses. Whereas, microorganic synthesis of PAA is impeded by the formation of by-product phenethyl alcohol due to quick, endogenous, and superfluous conversion of aldehydes to their corresponding alcohols, which resulted in less conversation of PAA from aldehydes. In this study, an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that does duty for a platform for aromatic aldehyde biosynthesis was used to prompt more PAA biosynthesis. We establish a microbial biosynthetic pathway for PAA production from the simple substrate phenylalanine in E. coli with heterologous coexpression of aminotransferase (ARO8), keto acid decarboxylase (KDC) and aldehyde dehydrogenase H (AldH) gene. It was found that PAA transformation yield was up to ~94% from phenylalanine in E. coli and there was no by-product phenethyl alcohol was detected. Our results reveal the high efficiency of the RARE strain for production of PAA and indicate the potential industrial applicability of this microbial platform for PAA biosynthesis.
| S-EPMC5445031 | biostudies-literature
Project description:BACKGROUND: Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate). RESULTS: Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity. CONCLUSIONS: This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.
| S-EPMC3616864 | biostudies-other
Project description:Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PAPERCLIP.
| S-EPMC4930481 | biostudies-literature
Project description:BackgroundCoumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.ResultsConstructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.ConclusionsTo produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.
| S-EPMC4419511 | biostudies-literature
Project description:BackgroundHydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource.ResultsWe synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D.ConclusionsUsing different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.
| S-EPMC5863376 | biostudies-literature
Project description:In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
2020-06-06 | GSE132139 | GEO
Project description:Hydrogen-dependent reduction of carbon dioxide to formic acid offers a promising route to greenhouse gas sequestration, carbon abatement technologies, hydrogen transport and storage, and the sustainable generation of renewable chemical feedstocks [1]. The most common approach to performing direct hydrogenation of CO2 to formate is to use chemical catalysts in homogeneous or heterogeneous reactions [2]. An alternative approach is to use the ability of living organisms to perform this reaction biologically. However, although CO2 fixation pathways are widely distributed in nature, only a few enzymes have been described that have the ability to perform the direct hydrogenation of CO2 [3-5]. The formate hydrogenlyase (FHL) enzyme from Escherichia coli normally oxidizes formic acid to carbon dioxide and couples that reaction directly to the reduction of protons to molecular hydrogen [6]. In this work, the reverse reaction of FHL is unlocked. It is established that FHL can operate as a highly efficient hydrogen-dependent carbon dioxide reductase when gaseous CO2 and H2 are placed under pressure (up to 10 bar). Using intact whole cells, the pressurized system was observed to rapidly convert 100% of gaseous CO2 to formic acid, and >500 mM formate was observed to accumulate in solution. Harnessing the reverse reaction has the potential to allow the versatile E. coli system to be employed as an exciting new carbon capture technology or as a cell factory dedicated to formic acid production, which is a commodity in itself as well as a feedstock for the synthesis of other valued chemicals.
| S-EPMC5772173 | biostudies-literature
Project description:Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food and induce protrusion of actin-rich membrane pedestals beneath themselves upon attachment to intestinal epithelia. EPEC then causes intestinal inflammation, diarrhea, and, among children, death. Here, we show that EPEC uses multiple tyrosine kinases for formation of pedestals, each of which is sufficient but not necessary. In particular, we show that Abl and Arg, members of the Abl family of tyrosine kinases, localize and are activated in pedestals. We also show that pyrido[2,3-d]pyrimidine (PD) compounds, which inhibit Abl, Arg, and related kinases, block pedestal formation. Finally, we show that Abl and Arg are sufficient for pedestal formation in the absence of other tyrosine kinase activity, but they are not necessary. Our results suggest that additional kinases that are sensitive to inhibition by PD also can suffice. Together, these results suggest that EPEC has evolved a mechanism to use any of several functionally redundant tyrosine kinases during pathogenesis, perhaps facilitating its capacity to infect different cell types. Moreover, PD compounds are being developed to treat cancers caused by dysregulated Abl. Our results raise the possibility that PD may be useful in treating EPEC infections, and because PD affects host and not bacterium, selecting resistant strains may be far less likely than with conventional antibiotics.
| S-EPMC491815 | biostudies-literature