Team:SCUT-China/Project/Chassis

Background

There are three enzymes needed in our chassis. Propionyl-CoA carboxylase(pcc), an enzyme needed in the biosynthesis of polyketides, is an enzyme complex containing two components playing the core role in our chassis, of which accA is the α biotinylated subunit functioning as the acetyl-CoA or propionyl-CoA carboxylase while pccB，the β subunit，acts as the carboxyl transferase【1】. BirA is a biotin ligase that improves the activity of accA, acts as an enhancer like sfp, the phosphopantetheinyl transferase facilitating posttranslational modification of the DEBS proteins in E.coli BL21(DE3) ^[2]. With the pcc and sfp genes transformed into E.coli BL21(DE3), it will express DEBS genes at the comparable level to S. erythraea or S.coelicolor ^[2].

Design

In E.coli, propionate is converted into propionyl-CoA and then utilized for metabolism with the steps catalyzed by proteins of the prp locus. Specifically, PrpE is in charge of the converting part, and PrpRBCD utilize propionyl-CoA as a metabolic substrate. Therefore, we delete prpRBCD genes in order that the ability of E.coli to utilize propionate as a carbon and energy source was eliminated. However, propionyl-CoA can only be produced through the converting reaction, so the prpE gene was placed under control of an IPTG-inducible T7 promoter.

By the way, using homologous recombination, a single copy of the sfp gene under control of the T7 RNA polymerase promoter was integrated in the prp operon of BL21(DE3), yielding E. coli BAP1.

The sfp gene product, which is part of the surfactin biosynthetic gene cluster in Bacillus subtilis, can effectively modify ACPs from all PKS subclasses as well as related peptidyl carrier protein and aryl carrier protein domains from nonribosomal peptide synthetases (NRPSs). Only with the function of sfp can propionyl-CoA be linked to ACPs, which is the basement of the reactions.

In the preparatory phase, we transformed pKD46 into BL21, as preparation for gene knock-out. pKD46 contains Red system which is composed from three genes (gam, bet, exo). The Gam protein prevents an E. coli nuclease of RecBCD from degrading linear DNA fragments, thus allowing preservation of transformed linear DNA in vivo. The bet gene product, Beta, is an ssDNA-binding protein that promotes annealing of two complementary DNA molecules, and the exo gene product, Exo, has a 5´ to 3´ dsDNA exonuclease activity.

After the preparation, we achieved the target gene as above by fusion PCR, which was then transformed into BL21(DE3), yielding E. coli BAP1. Using prpE and the fragment of prpR as homologous arm, we can ensure the gene replacement happens in targeted location. And Sh Ble gene product is zeocin-resistant, helping us to screen E.coli.

Result

After gene knock-out, we achieved BAP1 as the preparation for those following experiments. According to PCR results, prpRBCD gene had been knocked out while sfp gene and prpE gene is still kept on. Obviously, the gene had been transformed into the targeted location.

Reference

[1] Pfeifer B A, Admiraal S J, Gramajo H, et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli ［J］. Science,2001,291:1790
[2] M. Madyagol, H. Al-Alami, Z. Levarski, H. Drahovská, J. Tur˜na, S. Stuchlik, Gene replacement techniques for Escherichia coli genome modification, Folia Microbiol. 56 (2011) 253–263.
[3] A.R. Horswill, J.C. Escalante-Semerena, The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase, Microbiology 145 (1999) 1381–1388
[4] Pfeifer, B.A., Khosla, C., 2001. Biosynthesis of polyketides in heterologous hosts. Microbiol. Mol. Biol. Rev. 65, 106.