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

As the three enzymes are suitable to produce the substrate for all of our polyketide synthases(PKS), we decided to co-express them in one plasmid—the low copy plasmid pSB4A5 and make it the chassis for our expression of PKSs.

Figure 2: Our design of the desired genes on pSB4A5. Sfp was originally desigened to be knocked into the prp operon of E.coli BL21(DE3) by homologous recombination but was frame shifted, so we put it on pSB4A5 with individual PT7 and T7T. (A) The desired gene of chassis 1. (B) The desired gene of chassis 2. PT7, T7 promotor; RBS, ribosome binding site; 2TM, double T7terminator; birA, the biotin ligase gene; accA, the α biotinylated subunit gene of Propionyl-CoA carboxylase; pccB, the β subunit gene of Propionyl-CoA carboxylase; sfp, the phosphopantetheinyl transferase gene.

Here, we use T7 promoter and terminator to stay the same with our plasmid pSB1C3 carrying the genes encoding DEBS1 PKS so that they will be co-expressed under the same condition. RBS is placed between every two genes in order to ensure the transcription of every gene. Since the sfp gene knocked into the prp operon was frame shifted, we worried about whether it works as expected. Therefore，we constructed two chassis, of which one carries sfp(chassis 1) while another(chassis 2) doesn’t, and we will verify the activity of the sfp by transforming them into the E.coli BL21(DE3) separately with pSB1C3 carrying DEBS1 at the same time and see if there will be any difference between these two strain of E.coli BL21(DE3) under the same condition.

Construction

Since we didn’t have much time to buy Streptomyces coelicolor A3(2) to get gene accA and pccB from it by pcr. Besides, the Saccharopolyspora erythraea NRRL23338 we already had is not suitable to provide the two genes we needed[3], we chose to synthesize them. Meanwhile, we gained sfp and birA by pcr from E.coli BL21(DE3) and optimized their sequence by site-specific mutagenesis. Then, we constructed the pSB4A5 chassis by digest and assemble them step by step.

Result

1. Construction

Figure3: Analysis of the chassis we constructed. The 8.5kb plasmid pSB4A5 was digest by EcoRⅠand PstⅠinto two pieces, the 3.4kb backbone and the 5.2kb desired gene. M: 1kb marker; lane1、2 are parallel sample.

Figure 4:Analysis of the two devices transformed into E.coli BL21(DE3), the 15kb device 1 pSB1C3 and the 8.5kb device 2 pSB4A5. M1: 1kb marker; M2 DL15,000 marker; lane1~8: sample1~8 of E.coli BL21(DE3) transformed with both devices.

To test the transcription level of our desired genes, we did a series of RT PCR. Besides, as birA is an endogenous gene of E.coli BL21(DE3), we didn’t test it. From figure 5 we could draw a conclusion that accA, pccB and sfp were transcripted as expected.

Figure 5: Transcription level of accA、pccB and sfp, with 16S rRNA2 as the reference gene. ACC: accA; PCC: pccB; SFP: sfp.

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.