Identification of strong constitutive promoters in Burkholderia stagnalis TBRC 18363 for activating natural product production in Gram-negative bacteria

Gram-negative bacteria are emerging as an important source of natural products with pharmaceutical potential. However, the limited availability of genetic tools for drug discovery and sustainable production of secondary metabolites remains a challenge. Burkholderia spp. serve as a promising source for such tools, as these bacteria produce diverse natural products and are amenable to genetic modification. We sequenced the genome of Burkholderia stagnalis TBRC 18363 and performed transcriptomic analysis to identify genes highly expressed in early to late exponential cultures. We hypothesized that the sequences upstream of the most highly expressed genes contain strong and constitutive promoters active in heterologous Gram-negative hosts. Twenty-six B. stagnalis TBRC 18363 promoters were evaluated in Escherichia coli and Pseudomonas putida reporter systems. Two promoters, p2035 and p5642, exhibited superior performance in both systems. Promoter exchange experiments at biosynthetic gene clusters showed that these promoters can enhance the production titers of icosalide in B. stagnalis TBRC 18363 and FR900359, a G_q/11 protein inhibitor depsipeptide, in Chromobacterium vaccinii. Therefore, the p2035 and p5642 promoters are applicable for target gene overexpression in Gram-negative bacteria and can serve as tools for unlocking the potential of cryptic biosynthetic genes. KEY POINTS: • Strong constitutive promoters of Burkholderia stagnalis TBRC 18363 were identified. • Efficiencies of the selected promoters were evaluated in two heterologous hosts. • p2035 and p5642 promoters boosted BGC expression in Burkholderia and Chromobacterium.