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Part Collection

Part Collection

Part combination to raise the supply of precursor IPP and DMAPP

Isoprenoids are all derived from IPP and DMAPP, which are synthesized either by the MVA pathway or the MEP pathway. Therefore，the MVA and MEP pathways have been the targets of many metabolic engineering efforts to increase the supply of IPP and DMAPP in host microorganisms for improved terpenoid production.

In this study，we attempted to optimize the MEP pathway. It is well known that gene dxs plays an important role in enhancing IPP/DMAPP flux. Thus we need to over express the key enzyme Dxs.

It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. It was suggested that balanced activation of IspG and IspH could push the carbon flux away from not only HMBPP but also DXP, thus leading to a more efficient MEP pathway. The current researches show an appropriate ratio between ispG and ispH gene expression strength，and ispG expression strength can be increased to medium level and ispH expression strength to high level.

We attempted to construct plasmids with different copy numbers and promoters of different intensities by ispg and IspH in order to carry out the balanced activation.

The genes DXS and ispg are placed on the low copy pbbr1mcs-2 skeleton and the promoter is lac promoter of medium strength to construct pbbr1mcs-2-dxs-ispg (pmep-dg); the genes IspH, IDI and ISPA are placed on the high copy petduet-1 skeleton and the promoter is strong promoter T7 to construct petduet-1-1t7-isph-idi-ispa-yss (PET hiay).

In order to improve the supply of precursor IPP and dmapp in chassis cells, p35151 containing MVA pathway is combined with plasmid pmep-dg and pet hiay containing key enzyme gene of MEP and transfered into E.coli BL21 (DE3) to construct strain H5. We also combined p35151 with pet hiay, p35151, PMEP-DG and pET-HIAY to construct strains H3 and H4.

Strain name

Combination

Squalene yield（mg/L）

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 comparing squalene production, we found that on the basis of idi expressed genes and ispA, only expressing genes ispH will force its squalene production to decline. We speculate that this is because the overexpressed gene ispH consumes a lot of intermediate HMBPP. Also, the lack of gene ispG to express the upstream lead to the lack of HMBPP provided to ispH to react, thus hindering the MEP pathway in metabolic flow circulation;

By overexpressing Dxs and ispG, the squalene productivity has increased 4.3 times. We speculate that this is because the overexpressed gene ispG is constructed on the low-copy plasmid and was controlled by promoter of medium strength, thus it doesn’t produce much harmful intermediate HMBPP. However, the overexpression of Dxs promotes the downstream flow of metabolism, increasing the production of squalene.

Introducing the heterologous MVA pathway into E. coli had been widely used to increase the supply of IPP and DMAPP. Therefore, we also modified and optimized p35151 with the MVA pathway, dividing the 7 MVA pathway genes that contained in p35151 into upstream and downstream parts, and constructed them on the low-copy plasmid backbone.PMVA1 and pMVA2 were constructed with lac promoter of medium strength.

The modified pMVA1 and pMVA2 were combined with pET-YSS or pET-IAY respectively to co-transform BL21(DE3) and to construct strains H6 and H7

Strain name

Combination

Squalene yield（mg/L）

H1

p35151/pET-YSS

18.9

H6

pMVA1/pMVA2/ pET-YSS

617.8

H7

pMVA1/pMVA2/ pET-IAY

974.3

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.

Since chassis cells BL21(DE3) was replaced 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.

p35151, pMEP-DG and pUC-IAY were combined co-transformed E. coli XL1-blue to construct strain XH3, whose squalene production was 293.7mg/L.

When p35151, pMEP-DG and pUC-HIAY were combined to construct strain XH4, the yield of squalene was 504.0mg/L, 1.7 times higher than that of strain XH3, indicating that the composite parts containing Dxs, IspG and IspH we constructed could be applied to different E. coli chassis cells to optimize the MEP pathway and improve the supply of precursor IPP and DMAPP in the chassis cells.

Strain name

Combination

Squalene yield（mg/L）

XH3

p35151/ pMEP-DG/ pUC-IAY

293.7

XH4

p35151/pMEP-DG/ pUC-HIAY

504.0

XH5

pMVA1/pMVA2/ pUC-IAY

1274

When pMVA1, pMVA2 and pUC-IAY were combined together to co-transform E. coli XL1-blue, the squalene production was 1.3g/L, 1.3 times higher than that of the corresponding BL21 (DE3) strain H7, indicating that XL1-blue might be more suitable for being the chassis cell for the synthesis of squalene.