Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

Abstract

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

New Cyclodepsipeptide Derivatives Revealed by Genome Mining and Molecular Networking

“…To validate this hypothesis, the BGC coding for valinomycin biosynthesis was screened from a Streptomyces sp. CBMAI 2042 genomic library and the resulting pESAC13A derivative vector (PAC4H), containing the whole vlm gene cluster, was transferred by conjugation to S. coelicolor M1146 (Figure S5 and S6). The recombinant strain M1146/PAC4H#5 DSM 32578 (R. Sigrist, L. Gonzaga de Oliveira, 23 August 2017, Brazilian patent application BR1020170130239) reconstituted valinomycin and montanastatin production, supporting the hypothesis that both metabolites share a common biosynthetic origin.…”

High‐Performance Polymers from Nature: Catalytic Routes and Processes for Industry

“…To facilitate analysis of the role of SalE, and future manipulation of salinomycin biosynthesis, we undertook transplantation of the entire salinomycin gene cluster (Figures S18 and S19) as a single 136.8 kbp region of S. albus DNA into Streptomyces coelicolor M1154, a heterologous host strain producing no significant polyketide natural products of its own 17. A large‐insert P1‐based artificial chromosome (PAC) library in Escherichia coli was screened for the presence of the sal genes, and one positive PAC clone was selected, characterized and transferred by conjugation into S. coelicolor M1154 (Supporting Information and Figure S20) 18. HPLC‐MS analysis of the recombinant strain confirmed the production of authentic salinomycin at levels (≈2.4 mg mL −1 ) around 10 % of those typically seen from wild‐type S. albus (Figure 1).…”

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

“…Direct capture of BGCs and the use of transformation-associated- recombination (TAR) identified cosmomycin (Figure 2) and its analogues, and ammosamides A-C in the culture of recombinant S. coelicolor strains (Jordan and Moore, 2016; Larson et al, 2017). Following the cloning of gene cluster from S. tsukubaensis NRRL 18444 using a P1-derived artificial chromosome (PAC) expression vector, Bibb and his colleagues heterologously expressed the 83.5-kb FK506 gene cluster in a few S. coelicolor hosts (Jones et al, 2013); the yield of FK506 was increased from 1.2 mg/L to 5.5 mg/L in M1146 when the LuxR regulatory gene fkbN was over-expressed. Using the same PAC vector system, neoabyssomicin A/B and abyssomicin 2/4 were recently identified from a deep-sea isolate S. koyangensis SCSIO 5802 via expressing PAC constructs heterologously in M1152 (Tu et al, 2018).…”