Team:SEFLS Shanghai/Contribution

Contribution

IAY (rbs-idi-rbs-ispA-rbs-YSS) Composed By Three Genes idi、ispA and YSS and Relevant Rbs

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IAY can combine with optimized MEP and MYA pathway in various chassis cells, raises the yield of squalene greatly.

In E.coli BL21 (DE3) containing p35151, the production of squalene is 3.7 times higher when iay was introduced than that of YSS only; when iay is added with gene IspH, the production of squalene is 472.3mg/l, 25 times higher when it is combined with the optimized MEP pathway vector pmep-dg; when it is combined with the optimized MVA pathway vectors pmva1 and pmva2, the production of squalene is 974.3mg/l, compared with the strains containing pmva1, pmva2 and YSS, the yield of the strain is increased by 1.6 times, compared with that of the strain containing p35151 and 52 times with YSS.

After replacing the chassis cell with XL1-blue, iay is combined with pmva1 and pmva2, and the yield of squalene is 1.3g/l, 1.3 times higher than that of BL21 (DE3).

Yellow Carotenoid Synthesis Characterization

In order to find a suitable Squalene synthase, we cloned the four Squalene synthase genes from different origins, respectively KSS, NSS, YSS and thSQ & CrtN to the two multiple cloning sites on backbone pETDuet-1 and constructed the plasmid pETDuet-1T7-KSS-2T7-CrtN(pET-KN), pETDuet-1T7-NSS-2T7-CrtN(pET-NN), pETDuet-1T7-YSS-2T7-CrtN(pET-YN)和pETDuet-1T7-thSQS-2T7-CrtN(pET-thN).

To have the Metabolic flux flow towards FPP in the squalene, we overexpressed the genes idi and ispA and added them to plasmid pET-YN, yielding pET-IAY-CN.

Import those five plasmids into E. coli BL21(DE3) to detect the possible formation of carotenoid pigments. By testing the pigmentation level，We discovered that the pigmentation level in the expression of YSS is higher than other strains expressing NSS KSS or thSQS, showing that YSS is relatively active, while the pigmentation level in strains containing pET-IAY-CN is 2.2 times that of strains expressing pET-YN, demonstrating that we can significantly increase the metabolic flux towards FPP by expressing idi and ispA. Corresponding to the genes mentioned before are: Idi (Group: iGEM15_Nagahama (2015-05-31)_BBa_K1653002) IspA(BBa_K1653003 Group: iGEM15_Nagahama (2015-05-31))

Squalene Synthesis Characterization

We overexpressed idi and ispA in order to direct the metabolic flow to the squalene precursor FPP.

To improve the supply of precursors IPP and DMAPP, we first optimized the MEP pathway to overexpress its key enzymes Dxs, IspG and IspH. Genes DXS and ispG were placed on the skeleton of low-copy plasmid pbbr1mcs-2, and the promoter was lac of medium strength, in order to construct plasmid pbbr1mcs-2-dxs-ispg. Genes ispH, idi and ispA were placed on the skeleton of high-copy plasmid pETDuet-1, and the promoter was T7 of high strength, in order to construct plasimid pETDuet-1-1T7-Idi-IspA-Yss (pET-IAY) and pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).

Then pMEP-DG, pET-YSS, pET-IAY and pET-HIAY were transformed into E. coli BL21 (DE3) containing p35151. They are labelled as strain H1, H2, H3, H4 and H5. The strains H1 to H5 are fermented in shake flask at 30°C for 96 hours. The squalene yield of each strain is shown as follows:

Strain name

Containing plasmid

Squalene yield (mg/L)

H1

p35151/pET-YSS

18.9

H2

p35151/pET-IAY

69.3

H3

p35151/pET-HIAY

50.4

H4

p35151/pMEP-DG/ pET-IAY

298.2

H5

p35151/pMEP-DG/ pET-HIAY

472.3

After overexpressing gene Idi and IspA, the squalene yield of H2 was 3.7 times higher than that of H1 which only contained YSS. On the basis of the overexpression of gene Idi and IspA, the overexpression of only gene IspH decreased squalene yield. However, the squalene yield increased by 4.3 times after the overexpression of gene Dxs and IspG. If the gene Dxs, IspG and IspH were overexpressed at the same time, the squalene production was increased 6.8 times. This indicated that the squalene yield could be significantly increased by overexpressing the key enzyme genes Dxs, IspG and IspH to different extents in the MEP pathway.

Next, we also modified and optimized p35151 containing the MVA pathway, dividing 7 MVA pathway genes contained in p35151 into upstream and downstream parts and constructing them on low-copy plasmid backbone pBBR1MCS-1 and pBBR1MCS-2 whose promoters are medium-strength lac promoters. pBBR1MCS-1 has the same replicon as pBBR1MCS-2, so we replaced its replicon with p15A.

The modified pMVA1 and pMVA2 were respectively transformed into BL21(DE3) with pET-YSS or pET-IAY, and the squalene yield of the strains co-transformed into pMVA1, pMVA2 and pET-YSS was increased by 32.7 times than that of the strains co-transformed into p35151 and pET-YSS, indicating that the modified pMVA1 and pMVA2 significantly improved the supply of precursor IPP and DMAPP compared with p35151. The squalene production of the co-transformed pMVA1, pMVA2 and pET-IAY strains was 1.6 times higher than that of the co-transformed pMVA1, pMVA2 and pET-YSS strains, indicating that the overexpression of Idi and IspA increased the supply of precursor FPP, thus increasing the production of squalene.

We also tried to replace chassis cells, which is replacing BL21(DE3) with XL1-blue. To match with XL1-blue, we constructed the genes ispH, idi, ispA and YSS on pUC19m, which is a high-copy vector with a lac promoter, to construct pUC-IAY and pUC-HIAY.

E. coli XL1-blue co-transformed by p35151, pMEP-DG and pUC-HIAY, the squalene yield was 504.0mg/L after 96 hours of shake-flask fermentation, which was not significantly improved compared with the corresponding BL21 (DE3) strain. However, the yield of squalene was 1.3g/L when we used the co-transformed E. coli XL1-blue with pMVA1, pMVA2 and pUC-IAY. Compared with the corresponding strain of BL21 (DE3), the yield of the XL1-blue was increased by 1.3 times, indicating that XL11-blue might be a more chassis cell for the synthesis of squalene.