Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli
Fig 6
Complementation of the anthraquinone BGC with actinorhodin components.
(a) Typical exometabolome HPLC profiles of E. coli BL21, E. coli BL21 expressing empty vector pACYCDuet-1, KR antA knockout mutant pACYCAntΔAntA, wild-type Sco5086 KR complemented plasmid pACYCAntwtKR, refactored Sco5086 KR complemented plasmid pACYCAntrefKR, and pACYCAnthraquinone compared with fully characterised AQ256 standards and M9 growth media at 434 nm. The UV-Vis spectrum for peaks designated 1 are as follows: AQ256 standard λmax at 216, 264, 282, 434, and 583 nm; E. coli BL21 pACYCAnthraquinone λmax at 215, 263, 283, and 434 nm; E. coli pACYCAntrefKR λmax at 215, 283, and 435; and E. coli BL21 pACYCAntwtKR λmax at 216, 282, 435, and 585 in agreement with AQ256 (S15 Fig). Spectra were limited to 215–600 nm. (b) Typical exometabolome HPLC profiles of E. coli BL21, E. coli BL21 expressing empty vector pACYCDuet-1, ARO/CYC knockout mutant pACYCAntΔAntH, wild-type Sco5090 ARO/CYC complemented plasmid pACYCAntwtCYC, refactored Sco5090 ARO/CYC complemented plasmid pACYCAntrefCYC, and pACYCAnthraquinone compared with standards as above. Compound reference numbers are as described in Fig 2: 1: AQ256; 11: Aloesaponarin II; and 10: DMAC. The UV-Vis spectrum for the 3 most abundant peaks are as follows: 1: λmax at 216, 264, 282, 434, and 583 nm in agreement with AQ-256 (S15 Fig); 10: λmax at 217, 408, and 650 nm; 11: λmax at 214, 277, 409, and 582 nm, with tR of 16.004 min (960 s), 14.62 min (877.2 s), and 16.742 min (1004.5 s), respectively. (c) Typical exometabolite HPLC profile of E. coli BL21, E. coli BL21 expressing empty vector pACYCDuet-1, an α/β hydratase knockout mutant pACYCAntΔAntI, E. coli BL21 harbouring pACYCAnthraquinone, and anthraquinone standards. All numbering and λmax Figs are as in panel b. ARO, aromatase; BGC, biosynthesis gene cluster; CYC, cyclase; DMAC, 3,8-dihydroxy-methylanthraquinone carboxylic acid; HPLC, high resolution liquid chromatography; KR, ketoreductase; tR, retention time; UV-Vis, UV-visible.