Amit Kumar Jha

Enhanced Production of Nargenicin A 1 and Generation of Novel Glycosylated Derivatives

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.

Metabolic Engineering of Rational Screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L−1 and 129 mg L−1, which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L−1 and 27 mg L−1), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L−1 that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

Herboxidiene biosynthesis, production, and structural modifications: prospect for hybrids with related polyketide.

Herboxidiene is a polyketide with a diverse range of activities, including herbicidal, anti-cholesterol, and pre-mRNA splicing inhibitory effects. Thus, production of the compound on the industrial scale is in high demand, and various rational metabolic engineering approaches have been employed to enhance the yield. Directing the precursors and cofactors pool toward the production of polyketide compounds provides a rationale for developing a good host for polyketide production. Due to multiple promising biological activities, the production of a number of herboxidiene derivatives has been attempted in recent years in a search for the key to improve its potency and to introduce new activities. Structural diversification through combinatorial biosynthesis was attempted, utilizing the heterologous expression of substrate-flexible glucosyltransferase (GT) and cytochrome P450 in Streptomyces chromofuscus to generate structurally and functionally diverse derivatives of herboxidiene. The successful attempt confirmed that the strain was amenable to heterologous expression of foreign polyketide synthase (PKS) or post-PKS modification genes, providing the foundation for generating novel or hybrid polyketides.

Structural modification of herboxidiene by substrate-flexible cytochrome P450 and glycosyltransferase

Herboxidiene is a natural product produced by Streptomyces chromofuscus exhibiting herbicidal activity as well as antitumor properties. Using different substrate-flexible cytochrome P450s and glycosyltransferase, different novel derivatives of herboxidiene were generated with structural modifications by hydroxylation or epoxidation or conjugation with a glucose moiety. Moreover, two isomers of herboxidiene containing extra tetrahydrofuran or tetrahydropyran moiety in addition to the existing tetrahydropyran moiety were characterized. The hydroxylated products for both of these compounds were also isolated and characterized from S. chromofuscus PikC harboring pikC from the pikromycin gene cluster of Streptomyces venezuelae and S. chromofuscus EryF harboring eryF from the erythromycin gene cluster of Saccharopolyspora erythraea. The compounds generated were characterized by high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analyses. The evaluation of antibacterial activity against three Gram-positive bacteria, Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus, indicated that modification resulted in a transition from anticancer to antibacterial potency.

Genetic Manipulation of Nocardia Species.

Nocardia spp. are aerobic, Gram‐positive, catalase positive, and non‐motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities. It can be expected that knowledge of the biochemistry behind their pathogenicity will be crucial in developing effective treatment strategies. These genetic tools can be utilized to develop rational metabolic engineering approaches for crafting host strains with higher production or biotransformation ability.

Genetic evidence for the involvement of glycosyltransferase PdmQ and PdmS in biosynthesis of pradimicin from Actinomadura hibisca

Pradimicins are potent antifungal antibiotics with effective inhibitory effects against HIV-1. Pradimicin A consists of an unusual dihydrobenzo[α]naphthacenequinone aglycone substituted with a combination of D-alanine and two sugar moieties. Detailed genetic studies revealed most steps in pradimicin A biosynthesis, but the glycosylation mechanism remained inconclusive. The biosynthetic gene cluster of pradimicin A contains two putative glycosyltransferases, pdmQ and pdmS. However, the exact involvement of each gene in biosynthesis and the particular steps required for precise structural modification was unknown. In this study, the exact role of each gene was evaluated by insertional inactivation and complementation studies. Analysis of the metabolite from both of the disruption mutants revealed abolishment of pradimicin A and complementation resulted in the recovery of production. After deletion of pdmQ, pradimicin B was found to accumulate, whereas deletion of pdmS resulted in the accumulation of aglycone of pradimicin. Together, these results suggest that pdmS is responsible for the attachment of thomosamine to form pradimicin B which in turn is glycosylated by pdmQ to form pradimicin A. These results allowed us to deduce the exact order of terminal tailoring by glycosylation and provided insight into the mechanism of pradimicin A biosynthesis.

Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites

Saccharopolyspora spp. are aerobic, Gram‐positive, non‐acid‐fast, and non‐motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long‐term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp. Saccharopolyspora spinosa is a producer of spinosad, a prominent insecticide with selective activity against various insects.

Heterologous production of spectinomycin in Streptomyces venezuelae by exploiting the dTDP-d-desosamine pathway

Spectinomycin is an aminoglycoside antibiotic composed of actinamine and actinospectose, which are fused together by a putative glycosyltransferase, SpcG, during spectinomycin biosynthesis. Although previous studies have revealed the involvement of SpcA (myo-inositol monophosphatase), SpcB (dehydrogenase), SpcS2 (aminotransferase), and SpcM (methyltransferase) in the biosynthesis of actinamine, heterologous biosynthesis of spectinomycin via actinospectose has not been clearly elucidated. In this study, Streptomyces venezuelae was utilized as a source of dTDP-actinospectose from the pikromycin biosynthetic desosamine sugar pathway, and a recombinant vector, pSM5, carrying spcA, spcB, spcS2, spcM, and spcG was inserted into S. venezuelae. The formation of dTDP-spectinose was suspected through the use of dehydrogenase in the S. venezuelae chromosome. Herewith, the genetically engineered strain, S. venezuelae SM5, effectively produced up to 89.2mg/L in optimized medium. However, pSM5 in S. venezuelae YJ003, a dTDP-actinospectose-deficient strain, did not produce spectinomycin. This result demonstrates the use of a dTDP-actinospectose precursor produced in the desosamine pathway for heterologous production of spectinomycin in S. venezuelae.