Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli

Paul M D’Agostino,Tanja Woyke,Tobias A M Gulder,Thilo M Fuchs,Elke R Duell,Nicole Shapiro

doi:10.1186/s12934-019-1080-6

Abstract

BackgroundSerratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene.ResultsSequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA–D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling.ConclusionsGenome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

Highlights

Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways
Recent investigations in vivo and in vitro demonstrated that the S-adenosyl methionine (SAM)-dependent C-methyltransferase SodC is responsible for the addition of a methyl group to farnesyl pyrophosphate (FPP), and exhibits a surprising cyclisation activity leading to the phosphorylated version of pre-sodorifen, which is cyclized by the terpene cyclase SodD to yield the final bicyclic compound sodorifen (Fig. 1a) [13]
Within this study we present the application of direct pathway cloning (DiPaC) to functionally link a putative sodorifen biosynthetic gene cluster to sodorifen production in a recombinant host system and the optimization of the fermentative strategy to almost exclusive, high-level sodorifen production

Summary

Introduction

Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. Recent investigations in vivo and in vitro demonstrated that the SAM-dependent C-methyltransferase SodC is responsible for the addition of a methyl group to FPP, and exhibits a surprising cyclisation activity leading to the phosphorylated version of pre-sodorifen, which is cyclized by the terpene cyclase SodD to yield the final bicyclic compound sodorifen (Fig. 1a) [13]

Methods

Results

Discussion

Conclusion