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Putative catalytic mechanism of DBAT-catalysed hydrolysis of acetyl CoA and residues involved in catalytic process
Source publication
Taxoid 10β-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin...
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Objectives:
Taxoid 10β-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis.
Results:
Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activi...
Citations
... Compared with the conventional chemical methods, the enzyme conversion from 10-DAB to baccatin III has shown many superiorities, such as a stronger selectivity, higher efficiency and more eco-friendly [9][10][11]. Currently, research on enzymes that can catalyze the C10-hydroxyl acetylation has focused on the 10-deacetylbaccatin III-10-β-Oacetyltransferase (DBAT) enzymes from the Taxus plant, with the exception of a few enzymes derived from microorganisms. ...
Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.
... However, regarding the subsequent expression levels of hydroxylases, the expression levels of 16 T10αH genes were higher in TG. DBAT is another key enzyme that catalyzes 10-deacetylbaccatin III into baccatin III [47]. DBAT catalysis is a rate-limiting step in paclitaxel biosynthesis [48], and its expression level can affect the content of baccatin III, the last diterpene intermediate before paclitaxel [49]. ...
Paclitaxel is a potent anti-cancer drug that is mainly produced through semi-synthesis, which still requires plant materials as precursors. The content of paclitaxel and 10-deacetyl baccatin III (10-DAB) in Taxus yunnanensis has been found to differ from that of other Taxus species, but there is little research on the mechanism underlying the variation in paclitaxel content in T. yunnanensis of different provenances. In this experiment, the contents of taxoids and precursors in twigs between a high paclitaxel-yielding individual (TG) and a low paclitaxel-yielding individual (TD) of T. yunnanensis were compared, and comparative analyses of transcriptomes as well as chloroplast genomes were performed. High-performance liquid chromatography (HPLC) detection showed that 10-DAB and baccatin III contents in TG were 18 and 47 times those in TD, respectively. Transcriptomic analysis results indicated that genes encoding key enzymes in the paclitaxel biosynthesis pathway, such as taxane 10-β-hydroxylase (T10βH), 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT), and debenzoyl paclitaxel N-benzoyl transferase (DBTNBT), exhibited higher expression levels in TG. Additionally, qRT-PCR showed that the relative expression level of T10βH and DBAT in TG were 29 and 13 times those in TD, respectively. In addition, six putative transcription factors were identified that may be involved in paclitaxel biosynthesis from transcriptome data. Comparative analysis of plastid genomes showed that the TD chloroplast contained a duplicate of rps12, leading to a longer plastid genome length in TD relative to TG. Fifteen mutation hotspot regions were identified between the two plastid genomes that can serve as candidate DNA barcodes for identifying high-paclitaxel-yield individuals. This experiment provides insight into the difference in paclitaxel accumulation among different provenances of T. yunnanensis individuals.
... There were 13 mutational "hotspots" (Pro37, Val39, Asn42, Ile43, Ser122, His162, His123, Glu124, Ser159, Leu168, Gly171, Ile175, and Ser189) in the solvent channel (Figure 2) chosen for sitedirected mutagenesis. As the residue His162 was proved to be the key catalytic site of DBAT in our previous study (You et al., 2018). When the residue His162 was mutated to other amino acids, DBAT will completely lose its activity. ...
... Since the dbat gene was successfully cloned over two decades, research on DBAT has mainly focused on (i) cloning and heterologous expression of DBAT from different species (Guo et al., 2007;Han et al., 2014;Sah et al., 2019), (ii) the regional specificity and substrate diversity of DBAT (Loncaric et al., 2006(Loncaric et al., , 2007, and (iii) the exploration of the unnatural substrate (Li et al., 2017;Lin et al., 2018;You et al., 2018You et al., , 2019Huang et al., 2020). However, research on the thermal stability of DBAT has not been reported. ...
Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbat S189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.
... As the incidence of cancer increases, so does the commercial value of taxol [4]. However, the slow growth and extremely low taxol content of Taxus species lead to a fact that naturally extracted taxol is far from meeting the growing market demand [5]. Several alternative methods have been explored to increase taxol production in recent years, such as chemical total synthesis [6], in vitro Taxus cell culture [7], taxol-producing endophytic fungi [8], and heterogeneous synthesis [9], but most of them are difficult to commercialize due to their high cost and low production rate [10]. ...
Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. ‘Jinxishan’ (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3–7 years.
... Li et al. [72] generated a three-dimensional structure of DBAT and identified its active site using alanine scanning, and designed a double DBAT mutant (DBAT G38R/F301V) with a catalytic efficiency approximately six times higher than that of the DBAT wildtype (WT), which improved an in vitro one-pot conversion of 7-b-xylosyl-10-deacetyltaxol to taxol. Moreover, the activity essential residues of the enzyme DBAT, and the acylation mechanism from its natural substrate 10-DAB and acetyl CoA to baccatin III were investigated by You et al. [74]. Among them, residues H162, D166 and R363, located in the catalytic pocket of the enzyme, were important for DBAT activity; and residues S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG were important for acylation. ...
Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10β-hydroxylase (T10βOH) and a newly putative β-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.
... In our study, one unigene encoding TS and three unigenes encoding TAT were identified, and they predominantly expressed in T. yunnanensis. DBAT, another rate-limiting enzyme, catalyzes the formation of baccatin III from 10-deacetylbaccatin III [44]. BAPT is responsible for the transfer of a C13-side chain to baccatin III [45]. ...
Background
Taxol is an efficient anticancer drug accumulated in Taxus species. Pseudotaxus chienii is an important member of Taxaceae, however, the level of six taxoids in P. chienii is largely unknown.
Results
High accumulation of 10-DAB, taxol, and 7-E-PTX suggested that P. chienii is a good taxol-yielding species for large-scale cultivation. By the omics approaches, a total of 3,387 metabolites and 61,146 unigenes were detected and annotated. Compared with a representative Taxus tree ( Taxus yunnanensis ), most of the differentially accumulated metabolites and differential expressed genes were assigned into 10 primary and secondary metabolism pathways. Comparative analyses revealed the variations in the precursors and intermediate products of taxol biosynthesis between P. chienii and T. yunnanensis . Taxusin-like metabolites highly accumulated in P. chienii , suggesting a wider value of P. chienii in pharmaceutical industry.
Conclusions
In our study, the occurrence of taxoids in P. chienii was determined. The differential expression of key genes involved in the taxol biosynthesis pathway is the major cause of the differential accumulation of taxoids. Moreover, identification of a number of differentially expressed transcription factors provided more candidate regulators of taxol biosynthesis. Our study may help to reveal the differences between Pseudotaxus and Taxus trees, and promote resource utilization of the endangered and rarely studied P. chienii .
... They contributed equally to this work. process with recombinant DBAT (rDBAT) had provided a promising alternative for the enzymatic synthesis of baccatin III with relatively higher catalytic activity and cheaper substrates [10][11][12]. Yet, sustainability of rDBAT remains an issue. ...
... Glutaraldehyde, acetone, ethanol, n-butyl alcohol, isopropanol, acetonitrile, PEG1000, PEG4000, PEG6000, PEG8000, PEG12000, and ammonia sulfate were purchased from Guangzhou QiYun Biotech Ltd (Guangzhou, China). The Escherichia coli strain harboring DBAT gene was previously constructed and stored in our lab [11]. Cultivation was carried out in 250-mL Erlenmeyer flasks containing 100 mL of LB medium, incubated on a rotary shaker at 220 rpm and 37 °C for 3 h. ...
Taxoid 10β-O-acetyltransferase (DBAT) is the key enzyme to produce baccatin III, a key precursor in paclitaxel synthesis, by acetyl group transfer from acetyl-CoA to the C10 hydroxyl of 10-deacetylbaccatin III. In this study, the recombinant DBAT (rDBAT) was immobilized by cross-linked enzyme aggregates (CLEAs). To further optimize the enzyme recovery, single-factor experiment and response surface methodology were applied. 60% ammonium sulfate as precipitant, 0.05% glutaraldehyde as fixing agent, pH 7.0, 2 h as cross-linking time, 30 °C as cross-linking temperature were confirmed to be the optimum conditions to prepare the CLEAs-rDBAT in single-factor experiment. In addition, 62% for ammonium sulfate saturation, 0.15% for glutaraldehyde, and pH 6.75 were confirmed to be the optimum conditions with averagely 73.9% activity recovery in 3 replications, which was consistent with the prediction of response surface methodology. After cross-linking, the optimum temperature of CLEAs-rDBAT rose up to 70 °C and CLEAs-rDBAT could be recycled for three times.
Pharmacotherapy utility can be effectively mined from chemodiversity/biodiversity of Taxaceae- and Cephalotaxaceae-associated microbes. In this chapter, the research progress in biodiversity of Taxus-associated microbes is firstly summarized. Numerous endophytic fungi and endophytic bacteria are isolated and identified from T. media, T. mairei, T. baccata, T. wallichiana, and T. cuspidata. Rhizosphere microbes, phyllosphere microorganisms, and fungal pathogen are also isolated and characterized. The biodiversity of Cephalotaxus-, Torreya-, and Pseudotaxus-associated microbes is also striking. Endophytes, rhizosphere microbes, and pathogen of Cephalotaxus hainanensis, Cephalotaxus harringtonia, and Pseudotaxus are distinct. Taxane-producing microbes, isolated from non-Taxus plants, constitute an important bioprospecting achievement that cannot be ignored. Besides taxanes, various bioactive compounds have been isolated from the above microbes, for example, alkaloid, furan derivatives, terpenoids, aromatic compounds, acetogenins, coumarin and flavonoid, and anthraquinone. Multiple compounds of these microbes, including paclitaxel (PTX), had anticancer and cytotoxic activity. Some extracts/compounds also showed antifungal activity, antibacterial activity, antioxidant activity, antiinflammatory activity, antiparasitic activity, etc. The uses of metabolomics and transcriptomics unfold a beautiful picture to gain deeper insights into the microbial biosynthesis and physiological mechanism at the molecular level. Elicitor and strain improvement could help enhance the microbial production of medicinally important taxanes. Another important significance of microorganisms lies in recombinant expression and synthetic biology. Biosynthesis-related enzymes of PTX, for example, DBAT, PAM, CYPs, upstream enzyme, and diterpene synthase, have been heterologously expressed and characterized. PTX analog produced by recombinant expression and other recombinant enzymes have specific meanings, respectively. Metagenomics of T.×media, T. mairei, T. cuspidata, T. chinensis, and Torreya have been reported. Genomics and proteomics studies of Taxaceae- and Cephalotaxaceae-associated microbes should be strengthened for the sustainable utilization of these “small” and big resources.
