Combinatory Biosynthesis of Prenylated 4-Hydroxybenzoate Derivatives by Overexpression of the Substrate-Promiscuous Prenyltransferase XimB in Engineered E. coli

Abstract

Prenylated aromatic compounds are important intermediates in the biosynthesis of bioactive molecules such as 3-chromanols from plants, ubiquinones from prokaryotes and meroterpenoids from sponges. Biosynthesis of prenylated aromatic compounds using prokaryotic microorganisms has attracted increasing attention in the field of synthetic biology. In this study, we demonstrated that the production of 3-geranyl-4-hydroxybenzoic acid (GBA) and a variety of GBA analogues was feasible in a metabolically engineered E. coli by using XimB, a special prenyltransferase involved in the biosynthesis of xiamenmycin A in Streptomyces xiamenensis 318. XimB exhibits broad substrate specificity and can catalyze the transfer reaction of prenyl moieties with different carbon chain lengths to both the natural substrate 4-hydroxybenzoate (4-HBA) and to different substituted 4-HBA derivatives at C-2 and C-3. Feeding 4-HBA to an engineered E. coli equipped with a hybrid mevalonate pathway increased the production of GBA up to 94.30 mg/L. Considerable amounts of other GBA derivatives, compounds 4, 5, 6, 7, and 9, can be achieved by feeding precursors. The plug-and-play design for inserting C, C, and C prenyl diphosphate synthetases under the control of the T7 promoter resulted in targeted production of 3-dimethylallyl, 3-farnesyl-, and 3-geranylgeranyl-4-hydroxybenzoic acid, respectively. Furthermore, the valuable benzopyran xiamenmycin B was successfully produced in E. coli R7-MVA by coexpression of a complete biosynthetic gene cluster, which contains ximBDE.

Dynamical modelling of secondary metabolism and metabolic switches in Streptomyces xiamenensis 318

“…In a recent publication, we demonstrated that xiamenmycin B, an intermediate precursor of xiamenmycin A and one that does not incorporate threonine into the benzopyran core skeleton, could be synthesized in E. coli . [31]. Whether the surrogate E. coli host is more adaptable to the acetyl-CoA and NADPH stress than the natural Streptomyces host merits further study.…”

Constructing Microbial Hosts for the Production of Benzoheterocyclic Derivatives

“…Xiamenmycin B was detected with a product yield of 90.7 mg/L (Figure A, Table ). Given that XimB catalyzes the formation of prenylated products ( 1 , 2 , 3 , 4 , 5 , and 6 ) (Figure S2) from 4-hydroxybenzoic acid analogues, six 4-hydroxybenzoic acid analogues ( 7 , 8 , 9 , 10 , 11 , and 12 ) (Table , Figure S3) were separately fed at a final concentration of 100 mg/L into the 24 h old cultures of strain XL01. Benzopyran products 13 and 14 were detected by feeding 7 and 8 , respectively, as confirmed by UPLC-QTOF-MS, and their yields were 48.9 and 27.7 mg/L, respectively (Figure A, S4, S5, and Table ).…”

“…Angular FC (20) and PC (21) were also detected when feeding the corresponding precursor, C8prenylated umbelliferone (18) (Figure 5D), and the titer of 21 reached 0.44 mg/L. We tried to coexpress the prenyltransferases, i.e., PcPT and PT2, with ximD and ximE in E. coli to directly use an easily accessible and cheap substrate, umbelliferone (16) ximD-ximE2 were then transformed into E. coli Rosetta (DE3) strain R-MVA containing pCDF-Duet-1-mvaE-mvaS-ERG8-EGR12-MVD1-idi, 29 respectively, generating two strains, XL10 and XL11. Umbelliferone ( 16) was fed to each strain at a final concentration of 100 mg/L.…”

“…UPLC-QTOF-MS and NMR methods were previously described. 29 Stability Analysis of Coumarins. To evaluate the stability of coumarins, the strains S. xiamenensis 318, E. coli BL21 (DE3) and Rosetta (DE3) were cultured for 24 h. The cells were harvested by centrifugation (8000 rpm, 10 min, 4 °C), resuspended in 15 mL of Tris-HCl buffer (20 mM Tris, pH 8.0) and lysed by ultrasonication on ice.…”

Biosynthesis of Biscognienyne B Involving a Cytochrome P450‐Dependent Alkynylation

“…This possibility was corroborated with the detection of 3 when A. oryzae NSAR1 was cultured with 2 (see Figure S5A). Until now, several UbiA-type 4-hydroxybenzoate prenyltransferases from prokaryotes and eukaryotes have been identified, and usually show broad tolerance to the isoprenoid substrate, [14] whereas BisJ, as well as XimB, [15] is the few examples that are able to accept dimethylallyl pyrophosphate (DMAPP) as the prenyl donor.…”