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• Synthase Screening
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Squalene production by E.coli

Squalene production by E.coli

Screening of highly active squalene synthase

Because the precursors of squalene synthesis, farnesyl diphosphates (FPP), are available in E. coli, and thus, the production of squalene in E. coli is achieved simply by expressing squalene synthase (SQS). In this study, in order to obtain squalene synthase with higher activity, we cloned the squalene synthase from Y. lipolytica (YSS), S. cerevisiae (NSS), B. subtilis (KSS), and the truncated squalene synthase from human being (thSQS). The membrane-binding domains of hSQS (30 residues of the N-terminus and 47 residues of the C-terminus) were truncated based on the previous report, while YSS, NSS and KSS were expressed as the full-length sequence.

It was reported that the dehydrosqualene desaturase CrtN from Staphylococcus aureus converts squalene into yellow carotenoid pigments (63), permitting the colorimetric detection of squalene produced in the cell. In order to screen out squalene synthase with high activity rapidly, the SQS genes and CrtN gene were sub-cloned into the MCS1 and MCS2 of plasmid pETDuet-1respectively, yielding pETDuet-1T7-YSS-2T7-CrtN(pET-YN), pETDuet-1T7-NSS-2T7-CrtN(pET-NN), pETDuet-1T7-KSS-2T7-CrtN(pET-KN), pETDuet-1T7-thSQS-2T7-CrtN(pET-thN).

Plasmids with different SQS genes and CrtN gene were transferred with plasmid p35151 (Figure. 1C) into E. coli BL21 (DE3), and strains N1 (P35151/ pET-KN), N2 (P35151/ pET-NN), N3(P35151/ pET-thN) and N4(P35151/ pET-YN) were constructed. After 48 hours of fermentation, carotenoid pigments detection (Figure. 1B) was conducted on these strains. The results showed the pigmentation level of strain N4 expressing YSS was higher than that of strains expressing NSS, KSS and thSQS, indicating that YSS from Lysergic yeast has higher activity. So we decided to use YSS to construct the squalene synthesis pathway in E. coli.

Figure.1 A. sequence structure of four plasmids B. carotenoid pigments detection C. sequence structure of plasmid 35151

The Effect of Temperature on Squalene Accumulation

Because five components (tHMGR, HMGS, MK, PMK, PMD) of the mevalonate pathway (P35151) used here originated from yeast, we sought to determine the optimal temperature for these enzymes. Strain N4 was cultured at 30℃ or 37℃ to compare the pigmentation level. The results show that the pigmentation level was very low at 37℃, indicating that the optimal temperature for these enzymes was 30℃. Therefore, we chose 30°C as the fermentation temperature of the strains.

Functional Expression of YSS

In order to synthesize squalene in E. coli, the YSS gene was sub-cloned into plasmid pETDuet-1, yielding pET-YSS. Co-transformation of the resultant plasmid pET-YSS with p35151 (strain H1) resulted in squalene production of 18.9 mg/L (Figure. 1B). In order to further confirm that the fermentation product is squalene, we also used GC-MS to analyze its structure.

Figure2. A. relationship between peak area and squalene concentration B. result of co-transformation C. GC-MS analysis

Efficient Squalene Production

We had proposed three different strategies for engineering an efficient E. coli producer of squalene

Increase in the supply of precursor FPP

In order to increase the supply of precursor FPP, we overexpressed the gene idi and ispA. Genes idi and ispA were added to the plasmid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L (strain H2), an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151(Figure. 3). This result indicated that the overexpression of idi and ispA increased the supply of precursor, and thus increasing the yield of squalene.

Increase in the supply of precursor IPP and DMAPP

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.

Optimization of MEP Pathway

In order to optimize MEP pathway, we overexpressed the key enzymes Dxs, IspG and IspH (Go to Design) in the MEP pathway. 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. We did balanced activation by constructing genes ispG and ispH into plasmids with different copies and promoters of different strength.

These genes were respectively constructed into plasmids with different copies. The genes dxs and ispG were placed on the backbone of low-copy plasmid pBBR1MCS-2, and the promoter was medium strength lac promoters, to construct plasmid pBBR1MCS-2-dxs-ispG (pMEP-DG). Genes ispH, idi, and ispA were placed on the backbone of high-copy plasmid pETDuet-1, and the promoter was strong promoter T7 to construct plasmid pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).

P35151 was combined with plasmids pMEP-DG and pET-HIAY containing the key MEP enzyme genes. They were then co-transferred into E. coli BL21 (DE3) to construct strain H5. We also combined p35151 with pET-HIAY, p35151, pMEP-DG and pET-IAY to construct strains H3 and H4.

Name of Strains

Combination

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

Figure.3 construction of strains H1, H2 & H3

Comparing the squalene yield, we found that on the basis of overexpressing genes idi and ispA, only overexpressing gene ispH will decrease the squalene yield. It was speculated that the decline was because the overexpressed gene ispH consumed large amounts of intermediates HMBPP, which led to an insufficient supply of HMBPP for IspH to react, thus hindering the metabolic flow in MEP pathway.

When Dxs and IspG were overexpressed, the squalene yield increased by 4.3 times. We speculated that the overexpressed gene ispG was constructed on low-copy plasmids and was controlled by promoters of medium strength, so it didn’t produce large amounts of harmful intermediate HMBPP. And overexpressing Dxs promoted the metabolic flow, increasing the squalene yield.

Meanwhile, after overexpressing Dxs, IspG and IspH, the squalene yield was 1.6 times higher than only overexpressing Dxs and IspG, which meant that different overexpression combination of IspG and IspH could lead to a more efficient MEP pathway, thus increasing the squalene yield.

Optimization of MVA Pathway

Plasmid P35151 includes 6 genes (AtoB, HMGS, tHMGR, MK, PMK and PMD) in the MVA pathway and gene idi. These seven genes were integrated into a single operon. We optimized according to the MVA pathway in the strains producing fannene in the report, introducing MVA pathway gene included in plasmid P35151 separated in two parts on two plasmids to construct pBBR1MCS-1(p15A)-AtoB-HMGS-tHMGR (pMVA1) and pBBR1MCS-2-MK-PMK-PMD-Idi (pMVA2).

pMVA1 containing the first three genes atoB, HMGS and tHMGR in the MVA pathway took the plasmid pBBR1MCS-1 as the skeleton carrier, the promoter was lac of medium strength, and the replicon was replaced by p15A. pMVA2 containing the last three genes in the MVA pathway and gene idi took the plasmid pBBR1MCS-2 as the skeleton carrier, the promoter was lac of medium strength, and the replicon was pBBR1 oriV. The seven genes on plasmid pMVA1 and pMVA2 all came from gene P35151.

Plasmids pMVA1 and pMVA2 were used to co-transform BL21 with pET-YSS or pET-IAY. Strains H6 (pMVA1/pMVA2/pET-YSS) and H7 (pMVA1/pMVA2/pET-IAY) were constructed. Strains H6 and H7 were shaken and fermented at 30℃ for 96h, and the average yield of squalene was 617.8 mg/L for strain H6，974.3 mg/L for strain H7, an approximately 52-fold increase compared to strain H1. The squalene yield of the combination of pMVA1, pMVA2 and pET-YSS was 32.7 times higher than that of the combination of P35151 and DMAPP, indicating that the combination pMVA1 and PMVA2 after reconstruction has effectively improve the supply of precursors IPP and DMAPP. The squalene yield of the combination of pMVA1, pMVA2 and pET-IAY was 1.6 times higher than that of the combination of pMVA1, pMVA2 and pET-YSS, indicating that the overexpression of Idi and IspA has improved the supply of precursors FPP, increasing the squalene yield.

Name of Strains

Combination

Squalene Yield (mg/L)

H6

pMVA1/pMVA2/ pET-YSS

617.8

H7

pMVA1/pMVA2/ pET-IAY

974.3

Figure.4 A&B. construction of strains H6 & H7 C. squalene yield comparison

Replacement of Chassis Cells

In the report whose squalene yield was 230 mg/L, the chassis cell used to produce squalene was E. coli XL1-Blue. Different host strain has influence on the synthesis of target products. As a result, we tried to replace chassis cell BL21 (DE3) with XL1-Blue to observe the effect of different chassis cells on squalene yield.

The current expression carrier promoted by T7 promoter need to be expressed only in strain BL21(DE3). If the chassis is replaced with XL1-Blue, we need to construct genes ispH, idi, ispA and YSS on the high-copy carrier pUC19m whose promoter is lac to construct plasmid pUC19m-Idi-IspA-YSS (pUC-IAY) and plasmid pUC19m-IspH-Idi-IspA-YSS (pUC-HIAY).

p35151, pMEP-DG and pUC-IAY were combined together to co-transform E. coli XL1-Blue to construct strain XH3. The squalene yield was 293.7mg/L. p35151, mMEP-DG and pUC-HIAY were combined together to construct strain XH4. The squalene yield was 504.0mg/L, which was 1.7 times higher than the yield of strain XH3. This indicated that the composite parts containing Dxs, IspG and IspH we constructed can be used in different E. coli chassis cells to optimize MEP pathway and the supply of precursor IPP and DMAPP in the chassis cells.

Name of Strains

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

pMVA1, pMVA2 and pUC-IAY were combined together to co-transform E. coli XL1-Blue. The squalene yield was 1.3g/L(Figure. 5), which was 1.3 times higher than strain H7 of chassis cell BL21 (DE3), showing that XL1-Blue was more suitable as the chassis cell to synthesize squalene. Also, this level of squalene production is 5.5-fold of the highest reported value (230 mg/L) for E. coli.

Figure 5. construction of strains XH3, XH4 & XH5 and squalene yield comparison