Improved AP-3 production through combined ARTP mutagenesis, fermentation optimization, and subsequent genome shuffling.

Ansamitocin (AP-3) is an ansamycins antibiotic isolated from Actinosynnema pretiosum and demonstrating high anti-tumor activity. To improve AP-3 production, the A. pretiosum ATCC 31565 strain was treated with atmospheric and room temperature plasma (ARTP). Four stable mutants were obtained by ARTP, of which the A. pretiosum L-40 mutant produced 242.9 mg/L AP-3, representing a 22.5% increase compared to the original wild type strain. With seed medium optimization, AP-3 production of mutant L-40 reached 307.8 mg/L; qRT-PCR analysis revealed that AP-3 biosynthesis-related gene expression was significantly up-regulated under optimized conditions. To further improve the AP-3 production, genome shuffling (GS) technology was used on the four A. pretiosum mutants by ARTP. After three rounds of GS combined with high-throughput screening, the genetically stable recombinant strain G3-96 was obtained. The production of AP-3 in the G3-96 strain was 410.1 mg/L in shake flask cultures, which was 44.5% higher than the L-40 production from the parental strain, and AP-3 was increased by 93.8% compared to the wild-type A. pretiosum. These results suggest that the combination of mutagenesis, seed medium optimization, and GS technology can effectively improve the AP-3 production capacity of A. pretiosum and provide an enabling methodology for AP-3 industrial production.

First Ring-Expanded Maytansin Lactone Accessed by a New Mutasynthetic Variant.

A multiblocked mutant strain (ΔAHBA and Δasm12, asm21) of Actinosynnema pretiosum, the producer of the highly toxic maytansinoid ansamitocin, has been used for the mutasynthetic production of new proansamitocin derivatives. The use of mutant strains that are blocked in the biosynthesis of an early building block as well as in the expression of two tailoring enzymes broadens the scope of chemo-biosynthetic access to new maytansinoids. Remarkably, a ring-expanded macrolactone derived from ansamitocin was created for the first time.

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.

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.

Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein-Linked Extender Units in Ansamitocin Biosynthesis.

A few acyltransferase (AT) domains of modular polyketide synthases (PKSs) recruit acyl carrier protein (ACP)-linked extender units with unusual C2 substituents to confer functionalities that are not available in coenzyme A (CoA)-linked ones. In this study, an AT specific for methoxymalonyl (MOM)-ACP in the third module of the ansamitocin PKS was structurally and biochemically characterized. The AT uses a conserved tryptophan residue at the entrance of the substrate binding tunnel to discriminate between different carriers. A W275R mutation switches its carrier specificity from the ACP to the CoA molecule. The acyl-AT complex structures clearly show that the MOM-ACP accepted by the AT has the 2S instead of the opposite 2R stereochemistry that is predicted according to the biosynthetic derivation from a d-glycolytic intermediate. Together, these results reveal the structural basis of ATs recognizing ACP-linked extender units in polyketide biosynthesis.

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.

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.

Status Quo in Antibody-Drug Conjugates - Can Glyco- Enzymes Solve the Current Challenges?

Over the last years, a novel class of anti-cancer drugs named antibody-drug conjugates (ADCs) has been developed. Due to their limited off-target toxicity but highly potent cytotoxicity at tumor sites, ADCs have proven to be a good alternative to ordinary cancer treatment, such as chemotherapy or combination therapy. Numerous enhancements in antibody-drug engineering led to highly potent tumor targeting drugs with a wide therapeutic window. Two ADCs (Brentuximab vedotin and Trastuzumab emtansine) are already on the market and many others are in clinical trials. However, unstable linkers, low drug potency and unwanted bystander-effects are only some of the drawbacks of ADCs. Enzymes used in combination with prodrugs happen to be a promising alternative. The glyco-enzyme horseradish peroxidase (HRP) has proven to activate the hormone indole-3-acetic acid (IAA) to a highly potent cytotoxic drug. This combination of IAA and HRP has been investigated for the use in strategies such as gene-directed enzyme prodrug therapy (GDEPT) and antibody-directed enzyme prodrug therapy (ADEPT). This article reviews the current state of research in ADC engineering and describes the potential major enhancements through use of glycoenzymes in combination with a prodrug.

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 70 mg/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 51.5 mg/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.

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 (13)C-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.

Nocardiopsis ansamitocini sp. nov., a new producer of ansamitocin P-3 of the genus Nocardiopsis.

An alkalitolerant actinomycete strain, designated EGI 80425T, capable of producing ansamitocin P-3, was isolated from a saline-alkali soil sample of Xinjiang province, north-west China, and subjected to a polyphasic taxonomic characterization. Strain EGI 80425T formed non-fragmented substrate mycelia and white aerial hyphae with long spore chains. Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and rhamnose as the major sugar. The major fatty acids were anteiso-C17 : 0, iso-C16 : 0 and C18 : 1ω9c. The predominant menaquinones were MK-10(H4), MK-10(H6), MK-10(H8) and MK-9(H4). The G+C content of the genomic DNA of strain EGI 80425T was 70.2 mol%. Strain EGI 80425T showed highest 16S rRNA gene sequence similarity to Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (96.44 %). Phylogenetic analysis showed that strain EGI 80425T clustered with the members of the genus Nocardiopsis. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain EGI 80425T represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis ansamitocini sp. nov. is proposed. The type strain is EGI 80425T ( = CGMCC 9969T = KCTC 39605T).

Improvement of ansamitocin P-3 production by Actinosynnema mirum with fructose as the sole carbon source.

Ansamitocin P-3 (AP-3) is an active and potent anti-tumor maytansinoid, which is usually produced by Actinosynnema spp. In this study, the effects of different carbon sources on biomass and AP-3 production by Actinosynnema mirum were investigated. The results showed great biomass production behavior of A. mirum in glucose medium comparatively to other carbon sources. Interestingly, when fructose was used as the sole carbon source, the highest yield of AP-3 was obtained, which was about fourfold than that of strain cultured in glucose after 168 h. Further analysis conducted in regard to better understanding of such observations in glucose and fructose defined media showed that fructose improves AP-3 production through the stimulation of the key genes of the secondary metabolism pathways. It was concluded that fructose could be a potential carbon source for cost-effective production of AP-3 from an industrial point of view.

Process optimization with alternative carbon sources and modulation of secondary metabolism for enhanced ansamitocin P-3 production in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3), synthesized by Actinosynnema pretiosum, is a microtubule disruptor with significant antitumor activity. Although efforts have been made for the study of ansamitocin biosynthetic gene clusters and its fermentation improvement, the yield and productivity of AP-3 are still limited. In this study, fructose was found to be more beneficial to AP-3 production than glucose, and the culture condition was optimized via single-factor experiments and response surface method. The AP-3 concentration in the Erlenmeyer flasks reached 144 mg/L with the optimized medium containing fructose 9.36 g/L, glycerol 26.79 g/L and soluble starch 3.03 g/L, increased by ninefold compared with that before optimization. The result of medium optimization showed that fructose was an important element for effective increase in AP-3 production. Transcription of genes involved in primary metabolism and ansamitocin biosynthetic pathway was investigated to elucidate metabolic responses of cell metabolism to the substitution of fructose for glucose. It was demonstrated that using fructose as the major carbon source could relieve glucose repression and therefore result in flux rearrangement in primary metabolism for better providing biosynthetic precursors and stimulating the secondary metabolism in A. pretiosum. The results obtained might be of particular benefit to further enhancement of ansamitocin productivity.

Preparation of thermocleavable conjugates based on ansamitocin and superparamagnetic nanostructured particles by a chemobiosynthetic approach.

A combination of mutasynthesis, precursor-directed biosynthesis and semisynthesis provides access to new ansamitocin derivatives including new nanostructured particle-drug conjugates. These conjugates are based on the toxin ansamitocin and superparamagnetic iron oxide-silica core shell particles. New ansamitocin derivatives that are functionalized either with alkynyl- or azido groups in the ester side chain at C-3 are attached to nanostructured iron oxide core-silica shell particles. Upon exposure to an oscillating electromagnetic field these conjugates heat up and the ansamitocin derivatives are released by a retro-Diels-Alder reaction. For example, one ansamitocin derivative exerts strong antiproliferative activity against various cancer cell lines in the lower nanomolar range while the corresponding nanostructured particle-drug conjugate is not toxic. Therefore, these new conjugates can serve as dormant toxins that can be employed simultaneously in hyperthermia and chemotherapy when external inductive heating is applied.

Enhancement of ansamitocin P-3 production in Actinosynnema pretiosum by a synergistic effect of glycerol and glucose.

Ansamitocin P-3 (AP-3), a secondary metabolite produced by Actinosynnema pretiosum, is well known for its extraordinary antitumor properties and is broadly utilized in clinical research. Through this work, we found, for the first time, that the combination of glucose and glycerol as a mixed carbon source is an appropriate approach for enhancing the production of AP-3 by A. pretiosum. The amount yielded was about threefold that obtained with glucose as the sole carbon source. In order to better understand the mechanisms that channel glycerol metabolism towards AP-3 production, the activities of some key enzymes such as glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, phosphoglucomutase (PGM), and fructose 1,6-bisphosphatase were assessed. The results showed that glycerol affects the production of AP-3 by increasing PGM activity. Furthermore, qRT-PCR analysis revealed that transcriptional levels of structural genes asm14 and asm24, and primary genes amir5189 and amir6327 were up-regulated in medium containing glycerol.

Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production.

The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.

Biosynthesis of 3,5-AHBA-derived natural products.

Covering: 1957 to 2011. 3-Amino-5-hydroxy benzoic acid (3,5-AHBA) is a precursor for a large group of natural products, including the family of naphthalenic and benzenic ansamycins, the unique saliniketals, and the family of mitomycins. This review covers the biosynthesis of AHBA-derived natural products from a molecular genetics, chemical, and biochemical perspectives, and 174 references are cited.

Dual carbamoylations on the polyketide and glycosyl moiety by asm21 result in extended ansamitocin biosynthesis.

Carbamoylation is one of the post-PKS modifications in ansamitocin biosynthesis. A novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-ß-D-glucosyl moiety was identified from the ansamitocin producer, Actinosynnema pretiosum. Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Three new derivatives without the backbone carbamoyl group were isolated from an asm21 mutant and characterized by NMR spectroscopy. Among them, 18-O-methyl-19-chloroproansamitocin was the major product and the preferred substrate for macrolactam C-7 carbamoylation by Asm21. However, Asm21 exhibited higher catalytic efficiency toward the glucosyl moiety. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo. This work represents the first biochemical characterization of an O-carbamoyltransferase performing dual actions on both a polyketide backbone and a glycosyl moiety during ansamitocin biosynthesis.

Enhanced production of ansamitocin P-3 by addition of Mg2+ in fermentation of Actinosynnema pretiosum.

The effect of divalent metal ions (i.e., Mn(2+), Mg(2+), Zn(2+), Cu(2+), and Co(2+)) on the production of anticancer ansamitocin P-3 (AP-3) by submerged cultures of Actinosynnema pretiosum in medium containing agro-industrial residues was investigated, and Mg(2+) was found to be the most effective. Under the optimal condition of Mg(2+) addition, the maximal AP-3 production titer reached 85 mg/L, which was 3.0-fold that of the control. The activities of methylmalonyl-CoA carboxyltransferase (MCT) and methylmalonyl-CoA mutase (MCM) were enhanced. The content of two precursors, malonyl-CoA and methylmalonyl-CoA, was lower than that of control. This work demonstrates that Mg(2+) addition is a simple and effective strategy for increasing AP-3 production through the regulation of enzyme activity and pools of precursors. The information obtained can be helpful to its efficient production on large scale.

N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.

Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. Asm10 also showed broad substrate flexibility toward other N-desmethyl-ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis, possibly through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified.