Team:SCUT-China/Project/Overview

Overview

This summer, our team developed a novel approach for creating customized polyketides that overcomes the above mentioned limitations by engineering synthetic Polyketide Synthetases (PKSs). PKS is "called" the assemble line of polyketide. PKSs are very complicated that contain several kinds of domains in one module. Each module catalyst is to extend, modify or terminate the polyketides chain.

First of all, we attempted to synthesize triketide lactone by engineering Polyketide Synthetase—DEBS1+TE. Based on fact that single domain can work independently[1], we assembled domain in some order, in order to create new module of PKSs that can synthesize new polyketides. We chose a part of PKSs of erythromycin and obtain its sequence DEBS1, DEBS2 and DEBS3 to begin our project[2]. We first standardized every domain from DEBS1 and TE domain from DEBS3, and then assembled the DEBS1+TE as its original sequence. At the same time, we exchanged the some domains with the same function to compare their efficiency. Further, the heterologous loading modules were selected to substitute AT-ACP which is the loading module of DEBS1 so as to produce different kinds of triketide lactones. In order to fulfill Programmed Polykitide Synthesis (PPS), we try our best to establish the database can provide the proposal and protocol for choosing the functional chassis and the standardized domains you need for synthesizing novel polyketides.

These biomolecules, polyketides, are synthesized from some simple build blocks compounds such as coenzyme A (acetyl-CoA), propionyl-CoA and methylmalonyl-CoA. The DEBS1+TE utilize propionic acid or propionate to begin the synthesis of polyketides chain. To make sure the substrate of building block is enough to produce polyketides, we first deleted the prp operon that is responsible for propionate catabolism in E.coli BL21(DE3), so that the catabolism of propionate in BL21(DE3) was destroyed. In this way, the ability of BAP1 to utilize propionate as a carbon and energy source was eliminated. The prp operon include prpRBCDE, but the prpE gene is thought to convert propionate into propionyl-CoA, so we just have to delete the prpRBCD[2]. After deleting prpRBCD, we engineered the pathway of (2S)-methylmalonyl-CoA biosynthesis, which is the another building blocks of polyketides chain. The propionyl CoA carboxylase (pcc) genes from S. coelicolor which can synthesize the correct isomer of methylmalonyl-CoA, are transfered into the BL21[2].
Besides, domain ACP require gene sfp posttranslation modification to transfer the polyketides chain between two modules. In the process of deleting the prpRBCD, we integral sfp into the position of prpRBCD[2].

[1]Chaitan Khosla, Shiven Kapur, and David E. Cane. Revisiting the Modularity of Modular Polyketide Synthases, Curr Opin Chem Biol. 2009 April ; 13(2): 135–143.
[2]Blaine A. Pfeifer et al.Biosynthesis of Complex Polyketides in a Metabolically Engineered Strain of E. Coli Science 2001 291: 1790-1792 .