Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli
Fig 3
Identification and expression of soluble KS/CLF heterodimers in E. coli.
(a and b) Phylogeny of KS and CLF sequences from a dataset of 58 characterised type II PKSs derived from the MiBIG repository, respectively. The clades representing canonical type II KS and CLF are denoted by red and yellow wedges, respectively. Both alignments include FabF sequences from S. avermitilis, E. coli, and B. subtilis. Red and yellow dots denote His6-AntE and AntD, respectively. This colouring is conserved throughout the figure. (c) Denaturing PAGE showing soluble protein extracted from E. coli BL21(DE3) (lane 1) and E. coli BL21(DE3) pBbA2k-plumPKS harbouring the mPKS from P. luminescens (lane 2) induced at 30°C. Lanes 3 and 4 mirror those of 1 and 2; however, they show soluble protein expressed when incubated at 20°C. (d) Denaturing PAGE gel of AntD and E purified by IMAC. Lane 1: protein flow through, lane 2: protein eluted at 50 mM imidazole, lane 3: 500 mM imidazole column wash. (e) Western blot of purified AntDE protein showing signal corresponding to a single AntE band. Lane 1: PAGE ladder, lane 2: purified AntDE protein, and lane 3: His-tagged mCherry (approximately 29 kDa) fusion protein as positive control. All numbers correspond to standard protein ladders and are defined in kDa. Theoretical size of His6-AntE and AntD is 42.43 kDa and 46.16 kDa, respectively. CLF, chain length factor; His, Histidine tag; IMAC, immobilised metal ion affinity chromatography; KS, ketosynthase; MiBIG, Minimum Information about a Biosynthetic Gene cluster; mPKS, minimal PKS; PKS, polyketide synthase.