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Best Basic Part

Best Basic Part

Our Best Basic Part is BBa_K3166000. It is an enzyme localized to the membrane of the endoplasmic reticulum.

Coding sequence of Squalene Synthetase from Yersinia lipolytica (YSS)

YSS catalyzes the head-to-head condensation of two molecules of farnesyl diphosphate (FPP) to biosynthesize squalene. Squalene is a common precursor for the synthesis of many bioactive compounds, such as sterols, cholesterol and terpenes. Because of its unique physical and chemical properties, squalene has many beneficial effects on human health and life.

Characterization

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

It has been reported that dehydrosqualene desaturase CrtN from Staphylococcus aureus can convert squalene into yellow carotenoids.Thus, the activity of squalene synthase from different sources can be quickly compared, so as to screen out the optimum squalene synthase, by cloning CrtN gene downstream of squalene synthase and measuring the amount of yellow carotenoids. We cloned YSS, NSS, KSS and thSQS genes into a plasmid bearing CrtN and co-transformed plasmids containing different squalene synthase genes and CrtN gene into BL21(DE3) with plasmid p35151 containing MVA pathway. Single colony strains were selected and cultured in a tube for 48 hours. The bacteria were collected for acetone extraction, and the absorbance of the extracted solution was determined at 470nm using 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 in 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 resulting plasmid pET-YSS with p35151 has a squalene production of 18.9 mg/L.

We overexpressed idi and ispA in order to route the metabolic flux to the squalene precursor FPP. We introduced 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 hypothesized 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 still we need to optimize the MEP pathway. It is well known that 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 IspG and IspH by cloning them into different plasmids.

The DXS and IspG genes were placed on the backbone of low-copy-number plasmid pBBR1MCS-2, and the promoter was lac promoter of medium strength. IspH was cloned into the high-copy-number plasmid pET-IAY with a strong promoter T7. By co-transforming the two constructed plasmids 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 overexpressing idi and ispA.

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

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

PMVA2 used plasmid pBBR1MCS-2 as the backbone Its promoter was lac promoter of medium strength and its replicon was pBBR1 oriV It contained the other 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 yield (230 mg/L) in E. coli.