Engineering of Burkholderia thailandensis strain E264 serves as a chassis for expression of complex specialized metabolites

Abstract

Heterologous expression is an indispensable approach to exploiting natural products from phylogenetically diverse microbial communities. In this study, we constructed a heterologous expression system based on strain Burkholderia thailandensis E264 by deleting efflux pump genes and screening constitutive strong promoters. The biosynthetic gene cluster (BGC) of disorazol from Sorangium cellulosum So ce12 was expressed successfully with this host, and the yield of its product, disorazol F2, rather than A1, was improved to 38.3 mg/L by promoter substitution and insertion. In addition to the disorazol gene cluster, the BGC of rhizoxin from Burkholderia rhizoxinica was also expressed efficiently, whereas no specific peak was detected when shuangdaolide BGC from Streptomyces sp. B59 was transformed into the host. This system provides another option to explore natural products from different phylogenetic taxa.

Engineered Biosynthesis of Complex Disorazol Polyketides in a Streamlined Burkholderia thailandensis

“…Burkholderia thailandensis strain E264 was purchased from German collection of microorganisms and cell cultures GmbH (DSMZ), and the markerless mutants were established in our previous study , E. coli WM3064 was employed as a conjugal donor .…”

“…In our previous study, the yield of disorazol F 2 had been improved to 38.3 mg/ L, which provided the possibility to obtain novel derivatives of disorazol by gene cluster engineering. 24 Here, we report that gene inactivation of domains and deletion of modules resulted in the production of three unnatural disorazols. This study not only facilitates further structure−activity relationship (SAR) studies of disorazol but also provides a good example for engineering of other trans-AT PKSs with potential pharmaceutical value.…”

“…25 In order to circumvent this problem, site-directed mutagenesis mediated by RedEx, 26 a method combining Redαβ mediated homologous recombination, ccdB counterselection, and exonuclease-mediated annealing to perform gene editing was employed to inactivate the dehydratase (DH), ketoreductase (KR), and methyltransferase (MT) domains of the disorazol A gene cluster to obtain unnatural products. The plasmids of p15A-genta-int-attP-3P-dis with DH1, DH4, DH5, DH6, DH7, KR1, KR7, or MT2 domain inactivated were constructed, and mutants verified by DNA sequencing (Figure S1) were transferred into strain E264ΔBAC::attB 24 (Tables S1 and S2). Inactivation of the individual DH1 domain responsible for dehydration to generate olefinic moieties of module 1 resulted in the production of compound 2, and inactivation of DH7 resulted in the production of compound 3.…”

“…The details of metabolite extraction, HPLC, and LC-MS analyses were reported in the previous study. 24 Briefly, overnight cultures of transformants in LB broth were transferred into M9 broth for 24 h before 2% XAD-16 resin was added, then the incubation was continued for another 48 h. The resin was harvested by filtration and resuspended with MeOH. After 2 h incubation, the methanol was removed by evaporation in vacuo, and residues were dissolved with 1 mL of methanol.…”

“…Burkholderia thailandensis strain E264 was purchased from German collection of microorganisms and cell cultures GmbH (DSMZ), and the markerless mutants were established in our previous study. 24 Escherichia coli GB05red, E. coli GB05-dir, E. coli GB05-dir-gyrA462, and GB05-red-gyrA462 are stored in our lab. 33,34 E. coli WM3064 was employed as a conjugal donor.…”