Team:SCUT-China/Project/PPS

1.What kind of polyketide synthase do we choose as our candidate?

Polyketide almost exist in every organisms but many of their synthesis processes are unclear^[2]. First thing we did is to search suitable PKS. Erythromycin PKS modular 6-deoxyerythronolide B synthase(DEBS) from Saccharopolyspora erythraea is a good candidate^[3]. It has served as a module system for researching to engineer PKSs and to explore their structural plasticity and substrate tolerance^[4] for long. DEBS, encoded by the gene eryA, are divided into three part including totally six module. DEBS1 which include three module, is the first part of DEBS^[5].

There are two types of docking domains, class 1 is from actinomycetesmucous bacteria while class 2 is from cyanobacteria mostly ^[3]. Class 1 DD is also in the form of dimer just like the polyketide synthase. At present, the class 2 DD is seen to be a genetic engineering tool because of its shorter length and the advantages of the tight combination, and it will has great application prospect for the deeper research.
Docking domain consist of the ACP-side docking domain (ACP DD) at the C-terminus of the upstream and the KS-side docking domain (KS DD) at the N-terminus of downstream. Two parts locate in the downstream of ACP and upstream of KS respectively, and lie in the different modules. Each DD has formed the specificity binding because it has specificity connections site. Namely each docking domain is unique in the whole polyketone synthesis way. This important feature is to ensure the connection between the module and the module does not have randomness, making the product uniquely.
ACP of class 2 DD is about 40 animo acids shorter than class 1, which cannot form a polymerization area. Having the similar length with class 1 DD, KS of class 2 is unable to form a spiral structure domain coiled coil due to its strong polarity. Therefore, the class 2 DD have no the dimer, even though all of its structures are the even number. There are two alpha helix exist in the upstream and downstream of the class 2 DD separately. KS has alpha A and alpha B at the head of domain, while ACP has alpha 1 and alpha 2 at the end (Figure. 2). In order to have a closer combination, the alpha helices between alpha A and alpha B or between alpha 1 and alpha 2 form a sharp turn (sharp bend). This structure makes class 2 DD different from class 1 ^[3][4].

Design

From the previous introduction, it is obvious that the Class 2 DD has more advantages, the shorter length, closer combination, clearer effect of regulating protein function and higher transfer efficiency in completing upper intermediate. Therefore, we selected the Class 2 Docking Domain as our part. Because of its specificity (what we mentioned above), the connections between different sets of ACP and KS are not the same. Typical PKS subunits are tightly homodimeric and contain between one and six modules each. They are thought to associate with other multienzyme subunits through contacts at their C and N termini to form the overall PKS complexes, For example, the 6-deoxyerythronolide B synthase (DEBS) which assembles the polyketide core of erythromycin A contains three multienzyme subunits DEBS 1, DEBS 2, and DEBS3 each housing two extension modules ^[1][5]. We get the interest sequence of six pairs of DDs (DD1, DD2, DD3, DD4, DD5, DD6) by sequencing synthesis and add RFC 23 to insert restriction enzyme cutting sizes（Figure 3). At last, we built them to pSB1C3 vector.

Result

Go through long time exploration effort, we have got the standard DDs that can be used in different connections of ACP and KS. All docking domain are class 2 in our team. Each set of docking domain, the ACP on top, the upstream interface for RFC 23, downstream interface for RFC 10, the following to KS, the upstream interface for RFC 10, and downstream interface for RFC 23. The following general has no linker. Go through long time exploration efforts to build, our team have built out the different modules by using the same docking domain, and in the upstream and downstream added for assembly and clone site standard interface fusion protein needed - RFC 23, it will eventually constructing to pSB1C3 vector for use.

Conclusion

In our initial design, we aimed to create the standard general DD that can connect different Module (get more information about Module, please click here). Now, we have got the standard biobricks of DD but have no strict verification. Then, we will continue to work hard to complete the part of the experiment.

Reference

[1]Broadhurst RW, Nietlispach D, Wheatcroft MP, Leadlay PF, Weissman KJ(2003) The structure of docking domains in modular polyketide synthases. Chem Biol 10: 723–731.
[2]Worthington, A. S., Hur, G. H., Meier, J. L., Cheng, Q., Moore, B. S.and Burkart, M. D. (2008) Probing the compatibility of type II ketosynthase-carrier protein partners,ChemBioChem 9, 2096 –2103
[3]onia JB, Todd WG, Frank EB Kevin AR, Janet LS, and David HS(2009)Structural Basis for Binding Specificity between Subclasses of Modular Polyketide Synthase Docking Domains. ACS Chem Biol Vol.4 No.1
[4]Jonathan RW, Sarah SS, Douglas AH, William CB, William HG,David HS and Janet LS(2013)Cyanobacterial Polyketide Synthase Docking Domains:A Tool for Engineering Natural Product Biosynthesis. Chem Biol 20:1340–1351.
[5][Tang, Y., Kim, C.-Y., Mathews, I. I., Cane, D. E. and Khosla, C. (2006)The 2.7-angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase,Proc. Natl. Acad. Sci. U.S.A. 103, 11124 -11129.