Part:BBa_K3166000

Squalene Synthase(Yarrowia lipolytica)

Squalene Synthase(SQS) is an enzyme localized to the membrane of the endoplasmic reticulum. SQS participates in the isoprenoid biosynthetic pathway, catalyzing a two-step reaction in which two identical molecules of farnesyl pyrophosphate (FPP) are converted into squalene, with the consumption of NADPH.

In our study, we aim to achieve squalene synthesis in E.coli, so at the beginning, We cloned YSS, NSS, KSS and thSQS.

It has been reported that dehydrosqualene desaturated CrtN from staphylococcus aureus can convert squalene into yellow carotenoids. By extracting and detecting yellow carotenoids, the activity of squalene synthase from different sources can be quickly compared, so as to screen out the highly active squalene synthase. We constructed genes YSS, NSS, KSS and thSQS on a plasmid with CrtN and co-transformed plasmids containing different squalene synthase genes and CrtN genes into BL21(DE3) with plasmid p35151 containing MVA pathway. Constructed strains were selected monoclonal and fermented in a tube for 48 hours. The bacteria were collected for acetone extraction, and the absorbance of the extracted solution was determined at 470nm with an ultraviolet spectrophotometer. Results showed that the strain containing YSS gene had the highest absorbance, so we decided to use YSS to construct the squalene synthesis pathway E. coli.

To construct squalene synthesis pathway 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 resulted in squalene production of 18.9 mg/L.

We overexpressed idi and ispA in order to direct the metabolic flow to the squalene precursor FPP. We introduce genes idi and ispA into plasimid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L, an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151.

We speculated that if the supply of precursors IPP and DMAPP is increased, the squalene yield can be further increased. 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. We have introduced the MVA pathway into the chassis cell, and now we need to optimize the MEP pathway. It is well known that gene dxs plays an important role in enhancing IPP/DMAPP flux. It was demonstrated that balanced activation of IspG and IspH could improve isoprenoids production. We are going to overexpress the key enzymes Dxs and activate the balance of gene ispG and ispH by constructing them into different plasmids.

The gene Dxs and ispG were placed on the skeleton of low-copy plasmid pBBR1MCS-2, and the promoter was lac promoter of medium strength. The gene ispH was added to the high copy plasmid pET-IAY with a strong promoter T7. By co-transforming the two constructed plamids and p35151 into E. coli BL21 (DE3), the yield of squalene was 472.3mg /L, which was 6.8 times higher than that of the strain only overexpressing idi and ispA.

Plasmid P35151 contains 7 genes in the MVA pathway: AtoB, HMGS, tHMGR, MK, PMK, PMD and idi. These seven genes were integrated into a single operon. We attempted to divide the MVA pathway genes contained in plasmid p35151 into two parts and constructed them on pBBR1MCS-1 and pBBR1MCS-2. The constructed plasmids were as follows:

PMVA1 took plasmid pBBR1MCS-1 as the skeleton carrier, and the promoter was lac promoter of medium strength. As pBBR1MCS-1 has the same replicator as pBBR1MCS-2 does, its replicator was replaced with p15A, which contained the first three genes of MVA pathway, atoB, HMGS and tHMGR.

PMVA2 took the plasmid pBBR1MCS-2 as the skeleton carrier, its promoter was lac promoter of medium strength, its replicator was pBBR1 oriV, and it contained the four MVA pathway genes MK, PMK, PMD and idi. All 7 genes on plasmid pMVA1 and pMVA2 were derived from plasmid p35151.

Co-transformation of the plasmid pET-IAY with pMVA1 and pMVA2 resulted in squalene production of 974.3 mg/L, an approximately 52-fold increase compared to BL21(DE3) harboring pET-YSS and p35151. This level of production is 4-fold of the highest reported value (230 mg/L) for E. coli.

Usage and Biology

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) BBa_K3166997, pETDuet-1T7-NSS-2T7-CrtN(pET-NN) BBa_K3166800, pETDuet-1T7-YSS-2T7-CrtN(pET-YN) BBa_K3166998 and pETDuet-1T7-thSQS-2T7-CrtN(pET-thN) BBa_K3166801.

1. Target genes YSS, NSS, KSS, thSQS and crtN were obtained;

High-fidelity DNA polymerase was used to conduct PCR on genes YSS, NSS, KSS, thSQS and crtN. PCR results were as follows. The gel of PCR products was recycled to obtain DNA.

2. Double enzyme digestion of plasmid pETDuet-1 at MCS1 site was performed, and skeleton vector was obtained after DNA gel recycling. The following figure shows plasmid vector and gene fragment after gel recycling.

3. Gibson assembly kit was used to connect the target fragments YSS, NSS, KSS and thSQS into the MCS1 site of petduet-1, respectively. The PCR results of bacterial solution were as follows:

4. Extract the plasmid of the verified positive transformants to prepare the connection of crtN gene next;

5. The plasmid extracted from positive transformants was digested with double enzyme digestion at MCS2 site and connected with crtN gene after the gel recycling. The following figure shows the plasmid fragment and gene crtN after enzyme digestion.

6. The monoclone grown after the connection and transformation was verified by PCR, and the results were as follows: the positive transformant was simultaneously connected to the target gene and crtN gene;

7. The plasmids pET-KN, pET-NN, pET-YN and pET-thN of the positive transformant were extracted. The extracted plasmid and p35151 were co-transformed into BL21 (DE3) competent cell.

8. Monoclone was selected for fermentation in TB medium, and cultured at 180 r/min, 30°C for 48h after the induction by adding 0.1mm IPTG.

9. Bacteria were collected 48h later for carotenoid extraction and detection.

10. The results showed that the activity of squalene synthase YSS was the highest.

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.