Team:SCUT-China/Project/Overview

Polyketide is a large family of structurally diverse compounds^[1]. It exhibits a wide range of biological and pharmacological activities, particularly the antimicrobials. As far as we know, many antibiotics are derived from polyketide such as methymycin, erythromycin, amphotericin and so on. Nowadays, reaserchers about polyketide synthesis are focusing on the whole gene cluster of original polyketide or combinatorial biology^[2]. However, we can’t use the each domain flexibly by combinatorial biological methods. The study of the whole gene cluster limits the exploring of the modularity of polyketide synthase. All these researches didn’t make good use of the modularity of polyketide synthase.

Programmed Polykitide Synthesis (PPS)

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. By standardzing the domains of different functions, we can design and synthesize structurally various polyketide what we need just like programming.

Triketide Lactone Synthesis (TKLS)

First of all, we attempted to synthesize triketide lactone by engineering Polyketide Synthetase—DEBS1+TE. Based on fact that single domain have independent function^[3], we assembled domains in some order to create new module of PKSs that can synthesize new polyketides^[4]. We chose a part of PKSs of erythromycin and obtain its sequence DEBS1, DEBS2 and DEBS3 to begin our project^[5][6]. We first standardized every domain from DEBS1 and TE domain from DEBS3, and then assembled the DEBS1+TE as its original sequence(figure1). 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^[7]. In order to fulfill Programmed Polykitide Synthesis (PPS), we try our best to establish the database which can provide the proposal and protocol for choosing the functional chassis and the standardized domains you need for synthesizing novel polyketides.

Figure1:We arrange the DEBS1 as its original sequence.

Functional Chassis

These biomolecules, polyketides, are synthesized from some simple build blocks compounds such as coenzyme A (acetyl-CoA), propionyl-CoA and methylmalonyl-CoA^[8]. The DEBS1+TE utilize propionic acid or propionate to start 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 DE3 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 block 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^[5]. Besides, domain ACP requires the gene sfp to transfer the polyketide chain between two modules as posttranslation modification^[5][9]. In the process of deleting the prpRBCD, we inserted sfp into the position of prpRBCD^[10].

Reference

[1]Gao, Limei, et al. "Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production." Microb Cell Fact 12 (2013): 77.
[2]Menzella, Hugo G., et al. "Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes." Nature biotechnology 23.9 (2005): 1171-1176.
[3]Khosla, Chaitan, Shiven Kapur, and David E. Cane. "Revisiting the modularity of modular polyketide synthases." Current opinion in chemical biology 13.2 (2009): 135-143.
[4]Bhan, Namita, Peng Xu, and Mattheos AG Koffas. "Pathway and protein engineering approaches to produce novel and commodity small molecules."Current opinion in biotechnology 24.6 (2013): 1137-1143.
[5]Crosby, John, and Matthew P. Crump. "The structural role of the carrier protein–active controller or passive carrier." Natural product reports 29.10 (2012): 1111-1137.
[6]Yan, John, et al. "Functional Modular Dissection of DEBS1-TE Changes Triketide Lactone Ratios and Provides Insight into Acyl Group Loading, Hydrolysis, and ACP Transfer." Biochemistry 51.46 (2012): 9333-9341.
[7]Menzella, Hugo G., John R. Carney, and Daniel V. Santi. "Rational design and assembly of synthetic trimodular polyketide synthases." Chemistry & biology 14.2 (2007): 143-151.
[8]Chen, Alice Y., et al. "Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase." Journal of the American Chemical Society 128.9 (2006): 3067-3074.
[9]Beld, Joris, et al. "The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life." Natural product reports 31.1 (2014): 61-108.
[10]Pfeifer, Blaine A., et al. "Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli." Science 291.5509 (2001): 1790-1792.