Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952

Abstract

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.

ABC transporter genes from Streptomyces ghanaensis moenomycin biosynthetic gene cluster: roles in antibiotic production and export

“…For example, weak export of the antitumor polyketide doxorubicin from S. peucetius cells has been recently shown to decrease the overall productivity of the strain (Malla et al 2010, Song et al 2011). A similar situation probably applies to many recombinant pathways (Ostash et al 2010a), effectively limiting the potential of combinatorial biosynthesis in drug development.…”

Systems and synthetic biology to elucidate secondary metabolite biosynthetic gene clusters encoded inStreptomycesgenomes

“…Another representative example is the comparative proteomics on the doxorubicin less-producing mutant strain of S. peucetius ATCC 27952. 212 To increase the precursor CoA pool to support the doxorubicin production, the pantothenate kinase gene (panK) of E. coli was introduced into S. peucetius, however, doxorubicin production was instead decreased unexpectedly. Comparative proteomics revealed global proteomic changes including the decreased expression level of efflux proteins DrrAB of panK-integrated S. peucetius.…”

An Insight into the “-Omics” Based Engineering of Streptomycetes for Secondary Metabolite Overproduction

“…These approaches can also be used for engineering secondary metabolite target genes to enhance antibiotic production. For example, an attempt was made to increase the coenzyme A (CoA) pool using the pantothenate kinase ( panK ) gene to enhance production of DXR in S. peucetius ATCC 27952; however, the opposite occurred due to increased aglycone polyketide ε -rhodomycinone (RHO) [89]. To understand these results in detail, 2D gel electrophoresis was used to show that the efflux protein DrrA was overexpressed, resulting in 9.4-fold higher DXR production than that of a panK integrated strain, which showed that the proteomic approach is quite useful for host development and understanding the physiology of antibiotic production.…”