Understanding Autologous Spliceostatin Transcriptional Regulation to Derive Parts for Heterologous Expression in a Burkholderia Bacterial Host

Abstract

Burkholderia β-Proteobacteria are emerging sources of natural products. We are interested in developing Burkholderia sp. FERM BP-3421 into a synthetic biology chassis to facilitate natural product discovery. FERM BP-3421 produces autologous spliceostatins on gram per liter scale. We reasoned that transcription factors and promoters that regulate spliceostatin biosynthesis would provide valuable parts for heterologous expression. Herein we demonstrate that fr9A encodes a pathway-specific transcriptional activator of spliceostatin biosynthesis. In-frame deletion of fr9A abolished spliceostatin production, which was restored by genetic complementation. Using transcriptomics and green fluorescent protein (GFP) reporter assays, we identified four fr9 promoters, three of which are activated by LuxR-type regulator Fr9A. We then constructed an Fr9A-regulated promoter system that was compared to benchmarks and effectively applied for GFP and capistruin lasso peptide expression in an optimized host background. Our findings enrich the genetic toolbox for optimizing heterologous expression and promoting the discovery and development of natural products from Burkholderia bacteria.

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

“…Further exploration of this class of molecules has revealed new derivatives, in particular from the ubiquitous soil bacteria Pseudomonas aeruginosa , Streptomyces platensis , and Burkholderia sp. (Zhao et al 2019), which can be induced to make gram amounts of these compounds per liter of culture (Adaikpoh et al 2023; Eustáquio et al 2016). At the same time, evolutionary studies have noted that ancestors of present day fungi including Saccharomyces sp.…”

Heterologous Biosynthesis of Complex Bacterial Natural Products in Burkholderia gladioli

“…Recently, Schlegelella brevitalea DSM 7029, – Burkholderia thailandensis E264, , Burkholderia sp. FERM BP-3421, , Burkholderia gladioli pv. agaricicola HKI0676, Burkholderia ambifaria BCC1105, and Paraburkholderia phytofirmans PsJN have been reported as heterologous hosts.…”

“…agaricicola HKI0676, Burkholderia ambifaria BCC1105, and Paraburkholderia phytofirmans PsJN have been reported as heterologous hosts. They can heterologously produce diverse NPs from various bacteria, such as PKs and NRPs epothilone, ,, vioprolide, myxochelin, disorazol, and spliceostatin, the lasso peptide capistruin and burhizin-23, and polyynes cepacin and caryoynencin . Thus, Burkholderiales demonstrate great potential as a heterologous host for expressing exogenous BGCs.…”