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Pokhrel, Anaya Raj,Chaudhary, Amit Kumar,Nguyen, Hue Thi,Dhakal, Dipesh,Le, Tuoi Thi,Shrestha, Anil,Liou, Kwangkyoung,Sohng, Jae Kyung Elsevier 2016 MICROBIOLOGICAL RESEARCH Vol.192 No.-
<P><B>Abstract</B></P> <P>The <I>dnrO</I> gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of <I>Streptomyces peucetius</I> ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. <I>S. peucetius</I> was found to contain no functional copy of <I>bldA</I>-tRNA while a detailed examination of <I>dnrO</I> codons reveals the presence of TTA codon, which is rarely encoded by <I>bldA</I>-tRNA. Therefore, for evaluating the role of <I>dnrO</I> in DNR production, multiple engineered strains of <I>S. peucetius</I> were generated by heterologously expressing <I>bldA, dnrO</I> and combination of <I>bldA</I> and <I>dnrO</I>. Using these strains, the effects of heterologously expressed <I>bldA</I> and overexpressed <I>dnrO</I> were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of <I>dnrO</I> and master regulator <I>dnrI</I>, was found to be elevated in <I>bldA</I> containing strain in comparison to <I>dnrO</I> overexpressed strain. The <I>bldA</I> containing strain produces 45.7% higher DNR than <I>bldA</I> deficient wild type strain from culture broth with OD<SUB>600</SUB> of 1.45 at 72h. Heterologous expression of <I>bldA</I>–tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Jha, Amit Kumar,Pokhrel, Anaya Raj,Chaudhary, Amit Kumar,Park, Seong-Whan,Cho, Wan Je,Sohng, Jae Kyung Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.10
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-Omethylated rhamnose which are derived from S-adenosyl-methionine. 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 $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), 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. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ 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.
Nguyen Thi Hue,Anaya Raj Pokhrel,Nguyen Thanh Chung,Jae Kyung Sohng 한국당과학회 2017 한국당과학회 학술대회 Vol.2017 No.01
Drug development research requires a large amount of target proteins. Screening of drug target often requires 13C- and 15N- labeled protein, and higher protein expression has great advantages for obtaining isotope-labeled proteins. Among many of the proteins, glycosyltransferases(GTs) are attractive biocatalysts in producing a series of important bioactive natural products. In case of kanamycin, kanF gene encodes the first glycosyltransferase which acts both as glucosyltransferase and N-acetyl-glucosaminyl-transferase in kanamycin biosynthetic pathway of Streptomyces kanamyceticus. The recombinant expression of kanF gene in E. coli BL21 (DE3), BL21pLysS and BL21-CodonPlus® (DE3)-RP under T7 promoter-based system showed the expression in insoluble rather than soluble form. Further analysis of the codons revealed that this gene includes number of rare codons which are difficult to be translated in E. coli. Due to variations in codon usage between E. coli and Streptomyces, with high free energy of secondary structure of mRNA, KanF was not expressed under various condition tested. Thus, using codon optimized gene based on codon usage of E. coli has helped to overcome this problem and led to soluble expression of protein. Thus obtained KanF was used for making in vitro reaction for glycosylation of 2-DOS and the product was confirmed to be 2′ -Deamino-2′-hydroxyparomamine by high resolution Q-TOF mass analysis.
Amit Kumar Jha,Anaya Raj Pokhrel,Amit Kumar Chaudhary,Seong-Whan Park,Wan Je Cho,송재경 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.10
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-adenosyl-methionine. 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.
Paired-termini Antisense RNA Mediated Inhibition of DoxR in Streptomyces peucetius ATCC 27952
Amit Kumar Chaudhary,Anaya Raj Pokhrel,Nguyen Thi Hue,유진철,송재경 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
Our previous study provided an insight into DoxR as a negative regulator of doxorubicin production in Streptomyces peucetius ATCC 27952. Streptomyces hosts are advantageous in terms of producing a number of pharmaceuticals in low titer. Antisense RNAs (asRNAs) silencing strategy acts as an alternative tool for metabolic engineering of microorganisms for construction of an efficient cell factory. In this study, a paired-termini antisense RNAs (PTasRNAs) silencing strategy was employed for inhibition of DoxR to enhance doxorubicin production. To continue this endeavor, we designed and constructed the piBR702 vector for the expression of PTasRNAs in monocistronic mode. Further, two variants of asRNA, adoxR and bdoxR were designed and cloned into piBR702. All the rDNAs were transformed into S. peucetius to generate engineered strains. The engineered strains, S. peucetius A and S. peucetius B produced enhanced titers of doxorubicin, daunorubicin, and ε-rhodomycinone; however, no such change was seen in S. peucetius AB. Moreover, RT-PCR analysis of doxR from S. peucetius A and S. peucetius B, together with the higher production from S. peucetius A, confirmed adoxR as a better asRNA than bdoxR. The reason behind this could be due to the simple secondary structure and low binding free energy of adoxR (-419 kcal/mol) than bdoxR (-358.8 kcal/mol). Our study demonstrated that antibiotic production was enhanced significantly by inhibiting DoxR, a negative regulator in S. peucetius using PTasRNAs. In addition, this study further provides an insight into PTasRNAs as an effective tool for gene silencing in Streptomyces and its use as an effective tool for metabolic engineering.