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Cholesterol Biosynthesis | Sigma-Aldrich
The bottom portion of mevalonate pathway of E. faecium VTCC-B-935 isolated in Vietnam was introduced into a engineered carotenogenic E. coli resulted in improvement of β-carotene biosynthesis. The pRSET-A expression vector performed better than pET22b(+). Addition of glycerol as carbon source enhanced E. coli cell growth and biosynthesis of β-carotene almost 3 folds.
pR-IEIBY contains β-carotene biosynthesis genes and idi. pET28-K1K2D contains genes encoding enzymes catalyze reactions from mevalonate to IPP.
Bile Acid Synthesis, Metabolism and Biological Functions
Where, pR-EIBY and pET22-IEIBY are pRSET-A and pET22b(+) containing multicistronic operon of five genes including idi from E. coli DH5α, crtE, crtI, crtB, and crtY from Pantoea ananatis, respectively. These two vector were used for biosynthesis of β-carotene in recombinant E. coli BL21(DE3). pET28-K1K2D is pET28a(+) containing multicistronic operon of three genes including mvaK1, mvaK2, and mvaD from Enterococcus faecium VTCC-B-935. This vector was used for conversion of mevalonate to IPP. Major component of the vectors and restriction sites used in the construction were indicated. Vector maps were drawn using Vector NTI software version 11.5.1.
In this case report, we first examine the difference of expression vector backbones. In our previous study, three genes including crtE, crtB, and crtI were cloned from Pantoea ananatis for lycopene biosynthesis, and idi was cloned from E. coli for better balancing of IPP . These four genes, together with crtY, were introduced into pRSET-A and pET22b(+) resulted in multicistronic operon vectors pR-IEIBY and pET22-iEIBY, respectively. Secondly, differ from other previous publications, another source of genes encode for the bottom mevalonate pathway enzymes including mvaK1, mvaK2, and mvaD were cloned from Enterococcus faecium VTCC- B-935 isolated in Vietnam and introduced into pET28 vector forming pET28-mvaK1K2D. Subsequently, this vector was transformed into a recombinant E. coli strain which has already contained another expression vector pR-IEIBY. Finally, four additional carbon sources were investigated for higher production of β-carotene using our recombinant system.
Bile Acid Synthesis and Utilization
Biosynthesis partway of β-carotene is indicated in Figure 1. DMAPP and IPP, the building blocks of carotenoid are synthesized via 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway which is autonomous in our chosen host E. coli. However, in theory the natural yield of these precursors is only sufficient for natural need of E. coli in normal growth conditions which is far less than that required once the organism is used as a microbial factory to produce β-carotene. To address this issue, addition of the exogenous mevalonate (MVA) pathway was shown as a reasonable strategy [6,12,18,20,21]. The MVA pathway is divided into two portions, the upper (from acetyl-CoA to MVA) and the lower (from MVA to DMAPP and IPP).
Natural E. coli harbors MEP pathway that enable biosynthesis of FPP from G3P and pyruvate, as well as IPP isomerase catalyze the two-way conversion of IPP and DMAPP, the precursors of carotenoid (yellow part). These precursors could also be synthesized by the mevalonate pathway which is exogenous to E. coli. In this design, the bottom portion of MVA pathway from mevalonate to IPP was recruited from E. faecium VTCC-B-935 isolated in Vietnam (green part). IPP is subsequently used as building blocks for the process of β-carotene synthesis including four steps catalyzed by exogenous crt genes derived from P. ananatis. For better balancing between the two isoforms IPP and DMAPP, in addition to the endogenous idi gene positioned in the genome of E. coli host, another copy of this gene was introduced into the carotenogenic vector for co-over expression with crt genes (blue part).
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