Improvement of pristinamycin I (PI) production in Streptomyces pristinaespiralis by metabolic engineering approaches

Abstract

Pristinamycin, produced by Streptomyces pristinaespiralis, which is a streptogramin-like antibiotic consisting of two chemically unrelated components: pristinamycin I (PI) and pristinamycin II (PII), shows potent activity against many antibiotic-resistant pathogens. However, so far pristinamycin production titers are still quite low, particularly those of PI. In this study, we constructed a PI single component producing strain by deleting the PII biosynthetic genes (snaE1 and snaE2). Then, two metabolic engineering approaches, including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster (BGC), were employed to improve PI production. The final engineered strain ΔPIIΔpapR3/PI produced a maximum PI level of 132 mg/L, with an approximately 2.4-fold higher than that of the parental strain S. pristinaespiralis HCCB10218. Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb “supercluster” containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC, these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination (TAR) system. Collectively, the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

“…Unexpectedly, the deletion of the pristinamycin II biosynthesis genes (snaE1 and snaE2) decreased the yield of pristinamycin I. Further characterization of this pathway is therefore required for the development of a more suitable refactoring strategy . Improvement of product yield through the manipulation of cluster DNA is an approach which may require more study, especially in cases where secondary metabolism is not fully characterized.…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…ascomyceticus, [70][71][72] avermectin using S. avermitilis, [73] and pristinamycin I (PI) and II (PII) using Streptomyces pristinaespiralis. [74,75] To solve the issue of low production yield of tacrolimus in the natural producer, Wang et al [67] first determined the intracellular response of S. tsukubaensis to exogenous feeding of four precursors known to promote the tacrolimus biosynthesis. Using a weighted correlation network analysis (WGCNA) on a dataset containing 93 different intracellular metabolites measured with gas chromatography (GC)-MS and LC-MS/MS, metabolites highly associated with the tacrolimus biosynthesis were identified.…”

“…MFA to identify potential bottlenecks in biosynthesis 43.2% [86,87] Identification of three genes for overexpression to increase fluxes through rate-limiting pathway [74,75] Strain engineering by introduction of extra copies of the BGC, deletion of repressors, and overexpression of activators…”

“…[78] As the native host S. pristinaespiralis suffers from low yields, additional copies of the two BGCs responsible for the production of PI and PII were introduced in addition to the deletion of repressors. [74,75] . The metabolic engineering efforts were strongly aided from previous findings of the regulatory cascade governing the biosynthesis of both PI and PII in S. pristinaespiralis.…”

“…Besides the 2.4-fold increased production of PI compared to the wild-type, interestingly, the mutant with only the PII BGC removed showed 20-40% lowered PI production, compared to the wild-type, revealing a possible role of PII as a coactivator or inducer of PI production. [75] Further characterization efforts are needed to unravel the regulatory mechanism behind this observation and to additionally engineer the strain accordingly.…”