Ansamitocin P-3 Research Articles

Combinatorial Biosynthesis of 3-O-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A.pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A.pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.

Microfluidic fabrication of photo-responsive Ansamitocin P-3 loaded liposomes for the treatment of breast cancer.

Ansamitocin P-3 (AP-3) is a promising anticancer agent. However, its low solubility has limited its biomedical applications. The preparation of liposomal formulations for the delivery of low solubility drugs using the microfluidic platform has attracted increasing attention in the pharmaceutical industry. In addition, photodynamic therapy (PDT) is a non-invasive and efficient strategy for the treatment of cancers, making photodynamic liposomes one of the most promising drug delivery systems (DDSs). In this study, a recently developed microfluidic device (swirl mixer) was used for the manufacturing of temperature-sensitive liposomes (TSL) that can be activated by near-infrared stimulation for the treatment of breast cancer. Changing the processing parameters of the microfluidic system allowed for optimizing the properties of the produced liposomes (e.g., particle size and size distribution). For the first time, the anticancer drug AP-3 and the photosensitizer indocyanine green (ICG) were encapsulated into TSL (AP-3/ICG@TSL) during microfluidic processing. The results show that AP-3/ICG@TSL are biocompatible and can significantly reduce the toxicity of AP-3 to normal tissues. After infrared laser irradiation, the heat generated from ICG not only resulted in the cancer cell toxicity, but also facilitated the release of AP-3 in tumor cells. AP-3/ICG@TSL with infrared laser irradiation was found to be able to significantly inhibit the growth of MCF-7 multicellular tumor spheroids (MCTSs) in vitro and MCF-7 tumors subcutaneously inoculated in nude mice as an in vivo model. In addition, it also showed no signs of damage to other organs. The current results demonstrated that the AP-3/ICG@TSL fabricated using the microfluidic swirl mixer is a promising DDS for breast cancer therapy.

Two strategies to improve the supply of PKS extender units for ansamitocin P-3 biosynthesis by CRISPR–Cas9

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is a potent antitumor agent. However, lack of efficient genome editing tools greatly hinders the AP-3 overproduction in A. pretiosum. To solve this problem, a tailor-made pCRISPR–Cas9apre system was developed from pCRISPR–Cas9 for increasing the accessibility of A. pretiosum to genetic engineering, by optimizing cas9 for the host codon preference and replacing pSG5 with pIJ101 replicon. Using pCRISPR–Cas9apre, five large-size gene clusters for putative competition pathway were individually deleted with homology-directed repair (HDR) and their effects on AP-3 yield were investigated. Especially, inactivation of T1PKS-15 increased AP-3 production by 27%, which was most likely due to the improved intracellular triacylglycerol (TAG) pool for essential precursor supply of AP-3 biosynthesis. To enhance a “glycolate” extender unit, two combined bidirectional promoters (BDPs) ermEp-kasOp and j23119p-kasOp were knocked into asm12-asm13 spacer in the center region of gene cluster, respectively, by pCRISPR–Cas9apre. It is shown that in the two engineered strains BDP-ek and BDP-jk, the gene transcription levels of asm13-17 were significantly upregulated to improve the methoxymalonyl-acyl carrier protein (MM-ACP) biosynthetic pathway and part of the post-PKS pathway. The AP-3 yields of BDP-ek and BDP-jk were finally increased by 30% and 50% compared to the parent strain L40. Both CRISPR–Cas9-mediated engineering strategies employed in this study contributed to the availability of AP-3 PKS extender units and paved the way for further metabolic engineering of ansamitocin overproduction.Graphical

Improvement of Biosynthetic Ansamitocin P-3 Production Based on Oxygen-Vector Screening and Metabonomics Analysis

A novel approach involving exogenous oxygen vectors was developed for improving the production of biosynthetic Ansamitocin P-3 (AP-3). Four types of oxygen vectors including soybean oil, n-dodecane, n-hexadecane, and Tween-80 were applied to explore the effect of exogenous oxygen vectors on AP-3 yield. It was observed that soybean oil exhibited a better ability for promoting AP-3 generation than the other three oxygen vectors. Based on the results of the single-factor experiment, response surface methodology was employed to obtain the optimal soybean oil addition method. The optimum soybean oil concentration was 0.52%, and the addition time was 50 h. Under this condition, the yield of AP-3 reached 106.04 mg/L, which was 49.48% higher than that of the control group without adding oxygen vectors. To further investigate the influence of dissolved oxygen on precious orange tufts actinomycetes variety A. pretiosum strain metabolism and AP-3 yield, metabolomics analysis was carried out by detecting strain intermediate metabolites at various stages under different dissolved oxygen levels. Moreover, differential metabolite screening and metabolic pathway enrichment analysis were combined to exploit the effect mechanism of soybean oil on AP-3 production. Results suggested that primary metabolic levels of the TCA cycle and amino acid metabolism increased with the increase in dissolved oxygen level, which was beneficial to the life activities of bacteria and the synthesis of secondary metabolic precursors, thus increasing the production of AP-3.

Increased yield of AP-3 by inactivation of asm25 in Actinosynnema pretiosum ssp. auranticum ATCC 31565.

Asamitocins are maytansinoids produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565 (A. pretiosum ATCC 31565), which have a structure similar to that of maytansine, therefore serving as a precursor of maytansine in the development of antibody-drug conjugates (ADCs). Currently, there are more than 20 known derivatives of ansamitocins, among which ansamitocin P-3 (AP-3) exhibits the highest antitumor activity. Despite its importance, the application of AP-3 is restricted by low yield, likely due to a substrate competition mechanism underlying the synthesis pathways of AP-3 and its byproducts. Given that N-demethylansamitocin P-3, the precursor of AP-3, is regulated by asm25 and asm10 to synthesize AGP-3 and AP-3, respectively, asm25 is predicted to be an inhibitory gene for AP-3 production. In this study, we inactivated asm25 in A. pretiosum ATCC 31565 by CRISPR-Cas9-guided gene editing. asm25 depletion resulted in a more than 2-fold increase in AP-3 yield. Surprisingly, the addition of isobutanol further improved AP-3 yield in the asm25 knockout strain by more than 6 times; in contrast, only a 1.53-fold increase was found in the WT strain under the parallel condition. Thus, we uncovered an unknown function of asm25 in AP-3 yield and identified asm25 as a promising target to enhance the large-scale industrial production of AP-3.

Ansamitocin P3-Loaded Gold-NanoCage Conjugated with Immune Checkpoint Inhibitor to Enhance Photo-Chemo-Thermal Maturation of Dendritic Cells for Hepatocellular Carcinoma.

Immunotherapy is a newly developed method for cancer treatment, but still generates limited response in partial patients for hepatocellular carcinoma (HCC) because the immunity cycle is limited by the tumor microenvironment (TME). Herein, we introduce multifunctional gold nanocages (AuNCs)-based nanocarriers with Ansamitocin P3 (AP3) loaded and anti-PDL1 binding (AP3-AuNCs-anti-PDL1) which can combine photothermal therapy, chemotherapeutic agent-triggered DCs maturation, and checkpoint immunotherapy in one platform. The AP3-AuNCs-anti-PDL1 using Avidin-biotin to bind anti-PDL1 on the surface of AP3-AuNCs showed specifically cellular targeting compared to AuNCs, which can increase the immune responses. The AP3-AuNCs+NIR-10 min exhibited the highly activated DCs maturation with two-fold higher than control+NIR, which can be attributed to the significant release of AP3. The results illustrated the synergistic effect of tumor-associated antigens (TAAs) and controlled AP3 release under near infrared (NIR) in triggering effective DCs maturation. Among them, AP3 release played the more important role than the TAAs under PTT in promoting T-cell activation. These results illustrate the promising potential of AuNCs-based nanocarriers combined with AP3 and the checkpoint inhibitors to strengthen the positive loop of immunity cycle.

Efflux identification and engineering for ansamitocin P-3 production in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3) exhibits potent biological activities against various tumor cells. As an important drug precursor, reliable supply of AP-3 is limited by low fermentation yield. Although different strategies have been implemented to improve AP-3 yield, few have investigated the impact of efflux on AP-3 production. In this study, AP-3 efflux genes were identified through combined analysis of two sets of transcriptomes. The production-based transcriptome was implemented to search for efflux genes highly expressed in response to AP-3 accumulation during the fermentation process, while the resistance-based transcriptome was designed to screen for genes actively expressed in response to the exogenous supplementation of AP-3. After comprehensive analysis of two transcriptomes, six efflux genes outside the ansamitocin BGC were identified. Among the six genes, individual deletion of APASM_2704, APASM_6861, APASM_3193, and APASM_2805 resulted in decreased AP-3 production, and alternative overexpression led to AP-3 yield increase from 264.6 to 302.4, 320.4, 330.6, and 320.6 mg/L, respectively. Surprisingly, APASM_2704 was found to be responsible for exportation of AP-3 and another macro-lactam antibiotic pretilactam. Furthermore, growth of APASM_2704, APASM_3193, or APASM_2805 overexpression mutants was obviously improved under 300 mg/L AP-3 supplementation. In summary, our study has identified AP-3 efflux genes outside the ansamitocin BGC by comparative transcriptomic analysis, and has shown that enhancing the transcription of transporter genes can improve AP-3 production, shedding light on strategies used for exporter screening and antibiotic production improvement. KEY POINTS: • AP-3-related efflux genes were identified by transcriptomic analysis. • Deletion of the identified efflux genes led in AP-3 yield decrease. • Overexpression of the efflux genes resulted in increased AP-3 production.

The Therapeutic Possibility of EBV-Positive Nasopharyngeal Carcinoma through Targeting Somatostatin Receptors

Nasopharyngeal carcinoma (NPC) is a complex epithelial malignancy caused by both internal and environmental factors. Among them, Epstein–Barr virus (EBV) is a definitive etiological factor. The current therapeutic regimen for NPC is far from ideal and recurrence is commonly seen in patients after conventional radio and/or chemotherapy. We have thereby set out to develop new treatments for this deadly disease. Having analyzed a number of nasopharyngeal tissue and cell samples, we found that the level of somatostatin receptors (SSTRs) was positively correlated with EBV infection. Further, SSTR2 is the dominant SSTR isoform expressed in the EBV-positive (EBV (+)) NPC cells, a potential target for precise delivery of therapeutic agents. Cytotoxic effects of small molecule drugs including ansamitocin P-3 (AP-3), camptothecin and colchicine were subsequently evaluated in NPC cells. We found that AP-3 exhibits its strong anti-NPC activities with the IC50 values being 0.07 nM in EBV (+) NPC C666-1 cells and 0.09 nM in EBV (-) NPC CNE2 cells. In the xenograft animal model, AP-3 further shows inhibitory effects on NPC growth. These findings indicated that AP-3’s activity is not related to EBV in NPC cells. Thus, we proposed that SSTR2- specific delivery of AP-3 via linking AP-3 to somatostatin is likely a viable option to treat the EBV (+) NPC patients.

The Potential Applications of the Potent Cytotoxic Ansamitocin P-3 in the Treatment of Gastric Carcinoma

Gastric carcinoma is a refractory disease that seriously threatens human health globally. Unfortunately, its mortality is still high among common cancers. In order to develop a new type of anti-gastric cancer drug and avoid side effects of traditional chemotheray, a new drug-targeting strategy was considered in our studies. We firstly selected certain classic cytotoxic compounds such as ansamitocin P-3 (AP-3), colchicine and paclitaxel and investigated their efficacy in the suppression of gastric cancer cell growth and tumor growth. We found that these compounds displayed their potent suppressive activities. Particularly, AP-3 showed its significant tumor suppression in xenograft mouse model, and was also observed with its severe side effects. The results supported that AP-3 itself most likely was not druggable, but might be applied for drug conjugates via coupling it to a drug delivery vehicle to enhance its anti-tumor efficacy and reduce its side effects.

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes.

In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.

The Antitumor Agent Ansamitocin P-3 Binds to Cell Division Protein FtsZ in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards β-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of β-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM_5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.

Metabolomics analysis of Actinosynnema pretiosum with improved AP-3 production by enhancing UDP-glucose biosynthesis

Ansamitocin P-3 (AP-3) shows strong anticancer effects and has used as a payload for antibody-drug conjugates. Our previous study have shown that although genetically engineered Actinosynnema pretiosum strains with enhanced UDP-glucose (UDPG) biosynthesis displayed improved AP-3 production compared to the wild-type strain, the increase in yield was far from meeting the industrial demand. In this study, comparative metabolomics analysis complemented with quantitative real-time PCR analysis was performed for the wild-type strain and two mutants (OpgmOugp, ΔzwfΔgnd) to identify possible metabolic bottlenecks and non-intuitive targets for further enhancement of AP-3 production. We observed that enhancing intracellular UDPG availability facilitated the accumulation of intracellular N-demethyl-AP-3 and AP-3, where the transporting of them outside the cell still needs to be developed. We also found that the UDPG biosynthesis was closely associated with the availability of fructose in the medium and a suitable fructose feeding strategy could promote the further improvement of AP-3 titer. In addition, pathway abundance analysis revealed that undesired fatty acid accumulation and down-regulation of amino acid metabolism may be unfavorable for ansamitocin biosynthesis in later stage of production. These results indicate that genetic modification of the UDPG biosynthetic pathways may have pleiotropic effects on AP-3 production. Efforts must be made to eliminate these newly identified metabolic bottlenecks to boost AP-3 production in A.pretiosum.

Metabolomic change and pathway profiling reveal enhanced ansamitocin P-3 production in Actinosynnema pretiosum with low organic nitrogen availability in culture medium.

Ansamitocin P-3 (AP-3), a 19-membered polyketide macrocyclic lactam, has potent antitumor activity. Our previous study showed that a relatively low organic nitrogen concentration in culture medium could significantly improve AP-3 production of Actinosynnema pretiosum. In the present study, we aimed to reveal the possible reasons for this improvement through metabolomic and gene transcriptional analytical methods. At the same time, a metabolic pathway profile based on metabolome data and pathway correlation information was performed to obtain a systematic view of the metabolic network modulations of A. pretiosum. Orthogonal partial least squares discriminant analysis showed that nine and eleven key metabolites directly associated with AP-3 production at growth phase and ansamitocin production phase, respectively. In-depth pathway analysis results highlighted that low organic nitrogen availability had significant impacts on central carbon metabolism and amino acid metabolic pathways of A. pretiosum and these metabolic responses were found to be beneficial to precursor supply and ansamitocin biosynthesis. Furthermore, real-time PCR results showed that the transcription of genes involved in precursor and ansamitocin biosynthetic pathways were remarkably upregulated under low organic nitrogen condition thus directing increased carbon flux toward ansamitocin biosynthesis. More importantly, the metabolic pathway analysis demonstrated a competitive relationship between fatty acid and AP-3 biosynthesis could significantly affect the accumulation of AP-3. Our findings provided new knowledge on the organic nitrogen metabolism and ansamitocin biosynthetic precursor in A. pretiosum and identified several important rate-limiting steps involved in ansamitocin biosynthesis thus providing a theoretical basis of further improvement in AP-3 production.

Rational approach to improve ansamitocin P‐3 production by integrating pathway engineering and substrate feeding in Actinosynnema pretiosum

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is an important antitumor agent for cancer treatment, but its market supply suffers from a low production titer. The role of AP-3 unusual glycolate unit supply on its biosynthesis was investigated in this work by overexpressing the responsible gene cluster asm13-17 in A. pretiosum (WT). As a result, the accumulation of AP-3 and its intermediate glyceryl-S-ACP in the asm13-17-overexpressed strain (Oasm13-17) versus WT was enhanced by 1.94 and 1.49-fold, respectively. To provide a higher supply of another precursor 3-amino-5-hydroxybenzoic acid, asmUdpg was also overexpressed in Oasm13-17 (Oasm13-17:asmUdpg), and an improved AP-3 titer of 680.5 mg/L was achieved in shake flasks. To further enhance the AP-3 titer, a rational fed-batch strategy was developed in bioreactor fermentation of Oasm13-17:asmUdpg; and by pulse feeding 15 g/L fructose and 1.64 g/L isobutanol at 60, 96, and 120 hr, the AP-3 production level reached 757.7 mg/L, which is much higher than ever reported in bioreactors. This work demonstrated that a rational approach combining precursor pathway engineering with substrate feeding was very effective in enhancing the AP-3 titer, and this enabling methodology would be helpful to industrial production of this eye-catching drug.

Identification and Engineering of Post-PKS Modification Bottlenecks for Ansamitocin P-3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280.

The type-I polyketide ansamitocin P-3 (AP-3) is a potent antitumor agent. Its production is most likely hampered by the required multiple substrate supplies and complicated post-PKS modifications in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. For titer improvement, gene ansa30, encoding for a glycosyltransferase competing for the N-demethyl-AP-3 (PND-3) intermediate for AP-3 biosynthesis, was initially inactivated. In the mutant NXJ-22, the AP-3 titer was increased by 66% along with an obvious accumulation of PND-3, indicating that the N-methylation is a rate-limiting step. Alternatively, when abundant upstream intermediate 19-chloroproansamitocin was fed into a PKS mutant, 3-O-acylation was further identified along with the N-methylation as the rate-limiting steps. Subsequent overexpression of N-methyltransferase gene asm10 in NXJ-22 resulted in a 93% increase of AP-3 and a corresponding 92% decrease of PND-3. Additional supplementation of L-methionine, the precursor for SAM biosynthesis, substantially decreased the accumulation of PND-3. In parallel, the 3-O-acylation bottleneck was relieved by feeding with L-valine to NXJ-22, resulting in a 126% increase of AP-3. Eventually, a combined asm10 overexpression and supplementation of L-methionine and L-valine resulted in a 5-fold increase of AP-3, from 42 ± 2 mg L-1 to 246 ± 6 mg L-1 , without any noticeable accumulation of PND-3.

Combination of traditional mutation and metabolic engineering to enhance ansamitocin P-3 production in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.

Proteomic studies on anti-tumor agent ansamitocin P-3 producer Actinosynnema pretiosum in response to ammonium and isobutanol.

Our previous work showed that the biosynthesis of ansamitocin P-3 (AP-3), an anti-tumor agent, by Actinosynnema pretiosum was depressed by ammonium but enhanced by isobutanol in the medium. Here we show proteomics analyses on A. pretiosum in different fermentation conditions with and without ammonium or isobutanol using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization, and linear ion trap quadrupole mass spectrometry. Pairwise comparison of repetitive 2-DE maps was performed to find differentially expressed spots, and eight proteins were identified as functionally annotated ones. Among these proteins, D-3-phosphoglycerate dehydrogenase (PGDH) and glyceraldehyde 3-phosphate dehydrogenase showed statistically significant up-regulation in ammonium vs. basic or isobutanol medium, while fatty acid synthetase, histidine-tRNA ligase, transposase, molecular chaperone GroEL, SAM-dependent methyltransferase, and Crp/Fnr family transcriptional regulator were overexpressed in ammonium vs. basic medium. Based on the 2-DE data, exogenous L-serine which could inhibit the PGDH activity was added to the cultures with isobutanol, and a lower AP-3 production was confirmed under 2.5mM serine addition (24 or 48h).

Effects of the Methylmalonyl-CoA Metabolic Pathway on Ansamitocin Production in Actinosynnema pretiosum.

Ansamitocins, which may have antitumor activity, are important secondary metabolites produced by Actinosynnema pretiosum sp. auranticum ATCC 31565. As one of the precursors for ansamitocin biosynthesis, methylmalonyl-CoA may be a critical metabolic node for secondary metabolism in A. pretiosum. In this study, we investigated two key enzymes related to the methylmalonyl-CoA metabolic pathway: methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC). For MCM, inactivation of the asm2277 gene (encoding the large subunit of MCM) resulted in 3-fold increase in ansamitocin P-3 (AP-3) production (reaching 70mg/L) compared with that in wild-type A. pretiosum. The three genes responsible for PCC were asm6390, encoding propionyl-CoA carboxylase beta chain, and asm6229 and asm6396, which encoded biotin carboxylases, respectively. Heterogeneous overexpression of the amir6390 gene alone and concurrent overexpression of amir6390 with both amir6396 and amir6229 were carried out, and the resulting engineered strains could produce AP-3 at levels that were 1.6-fold and 3-fold (28.3 and 51.5mg/L in flask culture, respectively) higher than that in the wild-type strain. These results suggested that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively increase ansamitocin production in A. pretiosum.

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Ansamitocins, produced by Actinosynnema pretiosum, are a group of maytansinoid antibiotics that block the assembly of tubulin into functional microtubules. The precursors of ansamitocin biosynthesis are generally derived from the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid cycle. In this study, central carbon flux distributions were analyzed by 13C-based flux analysis to reveal the contribution of individual central carbon metabolism pathways. To direct more carbon flux into ansamitocin biosynthesis, pentose phosphate (PP) pathway only and the combination of PP pathway and Entner-Doudoroff (ED) pathway were weakened, respectively. Ansamitocin P-3 (AP-3) productions by both kinds of pathways weakened mutant strains were significantly enhanced in chemically defined medium. In order to draw metabolic flux to the biosynthesis of ansamitocins more efficiently, heterologous phosphoglucomutase was subsequently overexpressed based on a mutant strain with combinational regulation of PP pathway and ED pathway. More fluxes were successfully directed into the UDP-glucose synthetic pathway and the AP-3 production was further improved in this case, reaching approximately 185 mg/L in fermentation medium. It was demonstrated that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively improve ansamitocin production by A. pretiosum, suggesting a promising role of metabolic strategy in improving secondary metabolite production.

Effects of modulation of pentose-phosphate pathway on biosynthesis of ansamitocins in Actinosynnema pretiosum

Ansamitocins, produced by Actinosynnema pretiosum, are a group of maytansinoid antibiotics that block the assembly of tubulin into functional microtubules. The precursors of ansamitocin biosynthesis are generally derived from the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid cycle. In this study, central carbon flux distributions were analyzed by 13C-based flux analysis to reveal the contribution of individual central carbon metabolism pathways. To direct more carbon flux into ansamitocin biosynthesis, pentose phosphate (PP) pathway only and the combination of PP pathway and Entner-Doudoroff (ED) pathway were weakened, respectively. Ansamitocin P-3 (AP-3) productions by both kinds of pathways weakened mutant strains were significantly enhanced in chemically defined medium. In order to draw metabolic flux to the biosynthesis of ansamitocins more efficiently, heterologous phosphoglucomutase was subsequently overexpressed based on a mutant strain with combinational regulation of PP pathway and ED pathway. More fluxes were successfully directed into the UDP-glucose synthetic pathway and the AP-3 production was further improved in this case, reaching approximately 185mg/L in fermentation medium. It was demonstrated that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively improve ansamitocin production by A. pretiosum, suggesting a promising role of metabolic strategy in improving secondary metabolite production.