Construction of an Escherichia coli cell factory to synthesize taxadien-5α-ol, the key precursor of anti-cancer drug paclitaxel
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ABSTRACT:
SUBMITTER: Wu Q
PROVIDER: S-EPMC10992617 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
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Project description:CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene-5α-ol (T5α-ol) from taxadiene in paclitaxel biosynthesis. Past attempts expressing CYP725A4 in heterologous hosts reported the formation of 5(12)-oxa-3(11)-cyclotaxane (OCT) and/or 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) instead of, or in addition to, T5α-ol. Here, we report that T5α-ol is produced as a minor product by Escherichia coli expressing both taxadiene synthase and CYP725A4. The major products were OCT and iso-OCT, while trace amounts of unidentified monooxygenated taxanes were also detected by gas chromatography-mass spectrometry. Since OCT and iso-OCT had not been found in nature, we tested the hypothesis that protein-protein interaction of CYP725A4 with redox partners, such as cytochrome P450 reductase (CPR) and cytochrome b5, may affect the products formed by CYP725A4, possibly favoring the formation of T5α-ol over OCT and iso-OCT. Our results show that coexpression of CYP725A4 with CPR from different organisms did not change the relative ratios of OCT, iso-OCT, and T5α-ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5α-ol formation over other products, we used our results to clarify conflicting claims in the literature and discuss other possible approaches to produce paclitaxel via metabolic and enzyme engineering.
| S-EPMC5839926 | biostudies-literature
Project description:Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from Spirulina involves complex processes, which has led to increasing interest in the biosynthesis of PCB in Escherichia coli. However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from Thermosynechococcus elongatus BP-1(HOT) and PCB: ferredoxin oxidoreductase from Synechocystis sp. PCC6803 (PcyAS). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HOT(F29W/K166D) and PcyAS(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HOT and PcyAS mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of hemB and hemH, resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.
| S-EPMC10968380 | biostudies-literature
Project description:The title compound, C(20)H(30)O(2), {systematic name: (4aR,6aS,7R,11aS,11bR)-4,4,7,11b-tetra-methyl-1,2,3,4,4a,5,6,6a,7,11,11a,11b-dodeca-hydro-phenanthro[3,2-b]furan-4a-ol}, is a cas-sane furan-oditerpene which was isolated from the roots of Caesalpinia pulcherrima. The absolute configurations at positions 4a, 6a, 7, 11a and 11b are R, S, R, S and R, respectively. The mol-ecule has four-fused rings consisting of three cyclo-hexane rings and one furan ring. The three cyclo-hexane rings are trans-fused. Two cyclo-hexane rings are in chair conformations, while the third is in an envelope conformation. In the crystal structure, the mol-ecules are linked by inter-molecular O-H⋯O hydrogen bonds into a zigzag chain along the a axis. A short O⋯O contact [3.0398 (14) Å] is also present.
| S-EPMC3007238 | biostudies-literature
Project description:The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine that is phosphorylated at the 1 and 4' positions. Expression of the Francisella novicida lipid A 1-phosphatase FnLpxE in E. coli results in dephosphorylation of the lipid A proximal unit. Coexpression of FnLpxE and the Rhizobium leguminosarum lipid A oxidase RlLpxQ in E. coli converts much of the proximal glucosamine to 2-amino-2-deoxygluconate. Expression of the F. novicida lipid A 4'-phosphatase FnLpxF in wild-type E. coli has no effect because FnLpxF cannot dephosphorylate hexaacylated lipid A. However, expression of FnLpxF in E. coli lpxM mutants, which synthesize pentaacylated lipid A lacking the secondary 3'-myristate chain, causes extensive 4'-dephosphorylation. Coexpression of FnLpxE and FnLpxF in lpxM mutants results in massive accumulation of lipid A species lacking both phosphate groups, and introduction of RlLpxQ generates phosphate-free lipid A variants containing 2-amino-2-deoxygluconate. The proposed lipid A structures were confirmed by electrospray ionization mass spectrometry. Strains with 4'-dephosphorylated lipid A display increased polymyxin resistance. Heptose-deficient mutants of E. coli lacking both the 1- and 4'-phosphate moieties are viable on plates but sensitive to CaCl(2). Our methods for reengineering lipid A structure may be useful for generating novel vaccines and adjuvants.
| S-EPMC2943570 | biostudies-literature
Project description:Shikimate is a key intermediate in high demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug and oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/l of shikimate in 7-l fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.
| S-EPMC8788726 | biostudies-literature
Project description:Anthranilate is a key platform chemical in high demand for synthesizing food ingredients, dyes, perfumes, crop protection compounds, pharmaceuticals, and plastics. Microbial-based anthranilate production strategies have been developed to overcome the unstable and expensive supply of anthranilate via chemical synthesis from non-renewable resources. Despite the reports of anthranilate biosynthesis in several engineered cells, the anthranilate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of anthranilate production. Using the previously constructed shikimate-overproducing E. coli strain, two genes (aroK and aroL) were complemented, and the trpD responsible for transferring the phosphoribosyl group to anthranilate was disrupted to facilitate anthranilate accumulation. The genes with negative effects on anthranilate biosynthesis, including pheA, tyrA, pabA, ubiC, entC, and trpR, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroE and tktA, were overexpressed to maximize glucose uptake and the intermediate flux. The rationally designed anthranilate-overproducing E. coli strain grown in an optimized medium produced approximately 4 g/L of anthranilate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for microbial-based anthranilate production will play a key role in complementing traditional chemical-based anthranilate production processes.
| S-EPMC10050376 | biostudies-literature
Project description:BackgroundThe tobacco leaf-derived cembratriene-ol exhibits anti-insect effects, but its content in plants is scarce. Cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure. Moreover, the titer of reported recombinant hosts producing cembratriene-ol was low and cannot be applied to industrial production.ResultsIn this study, Pantoea ananatis geranylgeranyl diphosphate synthase (CrtE) and Nicotiana tabacum cembratriene-ol synthase (CBTS) were heterologously expressed to synthsize the cembratriene-ol in Escherichia coli. Overexpression of cbts*, the 1-deoxy-D-xylulose 5-phosphate synthase gene dxs, and isopentenyl diphosphate isomerase gene idi promoted the production of cembratriene-ol. The cembratriene-ol titer was 1.53-folds higher than that of E. coli Z17 due to the systematic regulation of ggpps, cbts*, dxs, and idi expression. The production of cembratriene-ol was boosted via the overexpression of genes ispA, ispD, and ispF. The production level of cembratriene-ol in the optimal medium at 72 h was 8.55-folds higher than that before fermentation optimisation. The cembratriene-ol titer in the 15-L fermenter reached 371.2 mg L- 1, which was the highest titer reported.ConclusionIn this study, the production of cembratriene-ol in E. coli was significantly enhanced via systematic optimization. It was suggested that the recombinant E. coli producing cembratriene-ol constructed in this study has potential for industrial production and applications.
| S-EPMC9872381 | biostudies-literature
Project description:The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.
| S-EPMC7383974 | biostudies-literature
Project description:Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis. Taxadiene binding followed type-1 substrate patterns with an observed KD of 2.1 ± 0.4 μM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed Vmax of 30 ± 8 pmol/min/nmolCYP725A4 and a KM of 123 ± 52 μM. Additionally, molecular operating environment (MOE) modeling was performed in order to gain insight into the interactions of taxadiene with CYP725A4 active site. Taken together, we demonstrate the successful expression and purification of the functional membrane-bound plant CYP, CYP725A4, in E. coli.
| S-EPMC5741078 | biostudies-literature
Project description:NG2 protein-expressing oligodendrocyte progenitor cells (OPC) are a persisting and major glial cell population in the adult mammalian brain. Direct synaptic innervation of OPC by neurons throughout the brain together with their ability to sense neuronal network activity raises the question of additional physiological roles of OPC, supplementary to generating myelinating oligodendrocytes. In this study we investigated whether OPC express neuromodulatory factors, typically synthesized by other CNS cell types. Our results show that OPC express two well-characterized neuromodulatory proteins: Prostaglandin D2 synthase (PTGDS) and neuronal Pentraxin 2 (Nptx2/Narp). Expression levels of the enzyme PTGDS are influenced in cultured OPC by the NG2 intracellular region which can be released by cleavage and localizes to glial nuclei upon transfection. Furthermore PTGDS mRNA levels are reduced in OPC from NG2-KO mouse brain compared to WT cells after isolation by cell sorting and direct analysis. These results show that OPC can contribute to the expression of these proteins within the CNS and suggest PTGDS expression as a downstream target of NG2 signaling.
| S-EPMC4429067 | biostudies-literature