Team:SCUT-China/Project/Have A Try

6-deoxyerythronolide B (6dEB), the macrocyclic core of the antibiotic erythromycin, is a complex product synthesized through the action of a multifunctional polyketide synthase (PKS) by the soil bacterium S.erythraea ^[4].

Therefore, the enzyme system, which is in charge of synthesizing 6dEB, is also called deoxyerythronolide B synthase (DEBS). The DEBS involves three polypeptide chains, including DEBS1, DEBS2, DEBS3, which have independent functions ^[6]. Correspondingly, the gene which codes DEBS, eryA, is divided into three, including eryAⅠ, eryAⅡ, eryAⅢ.

From the article of Pfeifer in 2001^[4], it is feasible to synthesis and functional expression of the gene cluster eryA in E.coli, using the method which is mentioned in the article.

Now, let’s have a base understanding of DEBS ^[4].

DEBS1: containing Module 1 (KS-AT-KR-ACP) and Module 2 (KS-AT-KR-ACP)

DEBS2: containing Module 3 (KS-AT- ACP) and Module 4 (KS-AT-DH-ER-KR-ACP)

DEBS3: containing Module 5 (KS-AT-KR-ACP) and Module 6 (KS-AT-KR-ACP)

Besides, the whole PKS chain includes a Loading Domain on the N-terminus and a TE Domain (thioesterase) on the C-terminus.

From the previous description, we know that each polypeptide chain (DEBS) has two Modules, and each Module includes several domains, which have similar structure and function.

A module contains but not limits to three domains: KS, AT, ACP (please click here to get more information about domain). Each a module can catalyze an extension and the cyclization of a carbon chain ^[4].

Design:

According to the design of our project PPS, we cannot make sure that we will make each domain as a standard part accurately. Maybe we will make the mistake that the function of some domains lose as we cut some necessary base pairs which we do not know. Therefore, we need to express the whole DEBS in E.coli functionally ^[5].

We choose PCR amplification to obtain the target gene segments. Base on the data of eryA we get from NCBI (the whole length of eryA is nearly 33kb), we have to use three different plasmids pSB1C3, pSB1A3 and pSB3K3 to carry eryAⅠ(11kb), eryAⅡ(11kb), eryAⅢ(9kb) as the carrying capacity of plasmid is limited.

Later, we will introduce all the three rebuilt plasmids into the host E.coli BAP1 and induce the functional expression of DEBS. If our design is OK, we will get the same product comparing with the experiment group, which uses combined standard domains to synthesize 6dEB.

Operation:

First of all, we find the sequence of eryA gene from NCBI and complete the genome extraction of S.erythraea which will be used as the PCR template.

It seems that we will meet the challenge of PCR amplification for the GC-enriched (≥70%) and large DNA fragment (nearly 11kb).

The first time, we design the primer pairs commonly with RFC 10. Primer STAR ^[2] & GenSTAR are chosen as the DNA polymerase while 3% DMSO is added to decrease the annealing temperature.

However, we failed on the first try as we only got the 2kb fragment.

We tried again immediately by changing the PCR enzyme and conditions.

We made the length of primer pairs longer to lower the high GC content of eryA. In addition to this, we done explore experiments about different concentration of DMSO from null to 10%.

This time, we got nothing unfortunately.

Latter, we choose mutant-enriched PCR. Extending the length of our target segment, we redesigned the primer pairs without RFC 10. During this period of time, we tried Primer STAR, GenSTAR, PCR Mix and KOD PCR enzyme and compared three-step process with two-step process ^[1][3].

At last, we can see the 5kb segment from the Gel Electrophoresis, which still not what we wanted.

Finally, we had to give up the idea of amplification eryA gene through PCR.

It sounds a pity.

Reference

[1]Zhanghua He, Yang Wang, Bingyu Ye, Minglei Shi, Dong Wang, Qiusheng Fan, Fen Huang, and Zhihu Zhao. Reconstruction of Erythromycin Macrocyclic Lactone Synthesis Pathway in Escherichia coli [J]. Chinese Journal of Biotechnology, 2012, 28(2):222-232 [2]Lihua Zhang, Yang Wang, Zhanghua He, Xiaojie Liu, Minglei Shi, Jianyong He, Zhihu Zhao. Cloning and Expression of Polyketide Synthases Gene eryAⅢ of Saccharopolyspora erythraea in Escherichia coli [J]. Letters in Biotechnology, 2010 [3]Frey UH, Bachmann HS, Peters J, Siffert W. PCR-amplification of GC-rich regions:’ slowdown PCR’ [J]. Nat Protoc. 2008,3(8):1312-7 [4]Blaine A. Pfeifer, Suzanne J. Admiraal, Hugo Gramajo, David E. Cane, Chaitan Khosla. Biosythesis of Complex Polyketides in a Metabolically Engineered Strain of E.coli [J]. Science, 2001, 291(44): 1790-1792 [5]Hugo G. Menzella,Sarah J. Reisinger,Mark Welch,James T. Kealey,Jonathan Kennedy,Ralph Reid,Chau Q. Tran,Daniel V. Santi. Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster[J]. Journal of Industrial Microbiology & Biotechnology. 2006 (1) [6] Menzella HG, Reisinger SJ, Welch M, Kealey JT, Kennedy J, Reid R, Tran CQ, Santi DV. Redesign, Synthesis and Functional Expression of the 6-deoxyerythronolide B polyketide synthase gene cluster [J]. Society for Industrial Microbiology Biotechnology. 2006 Jan;33(1):22-8