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Biosynthesis of β-carotene in engineered E

Beta-carotene is a precursor in the biosynthesis of Vitamin A in the human body.

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Beta Carotene | Biosynthesis | Metabolic Pathway

This explains why a long research phase preceded the achievement of the proof-of-concept for Golden Rice. By the early 1990s, the data accumulated became encouraging enough for Profs Peter Beyer and Ingo Potrykus to gather forces and dare to tackle this feat. Their breakthrough showed that only two transgenes were required to turn Golden Rice into a reality (Ye et al., 2000). The first transgene encodes a plant phytoene synthase (PSY), which utilises the endogenously synthesised geranylgeranyl-diphosphate (GGPP) to form phytoene, a colourless carotene with a triene chromophore (Burkhardt et al., 1997). The second gene encodes a bacterial carotene desaturase (CRTI) that introduces conjugation by adding four double bonds. Between 1993 and 1999, collaborative research between Peter Beyer and Prof Peter Bramley (Royal Holloway College, UK), was funded through EU networks B102-CT-930400, B104-CT97-2077 and FAIR CT96 1633. Working with genetically modified tomatoes, Peter Bramley established the advantage of using a single phytoene desaturase gene (bacterial CRTI), rather than introducing multiple plant desaturases (Romer et al., 2000). The combined activity of PSY and CRTI leads to the formation of lycopene, which is a red compound, its colour stemming from its undecaene chromophore, as is well established in tomato fruit. However, lycopene has never been observed in any rice transformant and different genetic backgrounds. Instead, α- and β-carotene are found together with variable amounts of oxygenated carotenoids (xanthophylls), such as lutein and zeaxanthin. The carotenoid pattern observed in the grain's endosperm revealed that the pathway proceeded beyond the end point expected from the enzymatic action of the two transgenes alone. A detailed analysis of the underlying mechanism has been published (Schaub et al., 2005). The findings are explained in some detail below (Figure 2).

Figure 1: Biosynthesis pathways of β-carotene in recombinant E. coli engineered in this study.

" Synthesis of Coenzyme Q₁₀ and β-carotene by Yeasts Isolated from Antarctic Soil and Lichen in Response to Ultraviolet and Visible Radiations "

Mechanism of the Biosynthesis of Vitamin A from β-Carotene

AB - β-carotene hydroxylase is known to be involved in zeaxanthin synthesis. Disruption of the crtR gene encoding β-carotene hydroxylase in Synechocystis sp. PCC 6803 resulted in the absence of both zeaxanthin synthesis and myxoxanthophyll accumulation in the mutant strain. A new carotenoid was detected in this strain. Its chemical structure was close to that of myxoxanthophyll, but the hydroxyl group on the β-ring was lacking. This compound, deoxy-myxoxanthophyll, most likely is an intermediate in the myxoxanthophyll biosynthesis pathway. Therefore, β-carotene hydroxylase is involved not only in zeaxanthin synthesis but also in myxoxanthophyll synthesis in Synechocystis. Furthermore, myxoxanthophyll in Synechocystis was found to have a higher molecular mass than what was determined in other species. This difference is likely to be due to a difference in the sugar moiety. Copyright (C) 1999 Federation of European Biochemical Societies.

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).

The biosynthesis of cyclic carotenes

N2 - β-carotene hydroxylase is known to be involved in zeaxanthin synthesis. Disruption of the crtR gene encoding β-carotene hydroxylase in Synechocystis sp. PCC 6803 resulted in the absence of both zeaxanthin synthesis and myxoxanthophyll accumulation in the mutant strain. A new carotenoid was detected in this strain. Its chemical structure was close to that of myxoxanthophyll, but the hydroxyl group on the β-ring was lacking. This compound, deoxy-myxoxanthophyll, most likely is an intermediate in the myxoxanthophyll biosynthesis pathway. Therefore, β-carotene hydroxylase is involved not only in zeaxanthin synthesis but also in myxoxanthophyll synthesis in Synechocystis. Furthermore, myxoxanthophyll in Synechocystis was found to have a higher molecular mass than what was determined in other species. This difference is likely to be due to a difference in the sugar moiety. Copyright (C) 1999 Federation of European Biochemical Societies.

The explanation is that enzymes further down the pathway, such as lycopene cyclases (LCYs) and α- and β-carotene hydroxylases (HYDs), are still being produced in wild-type rice endosperm, while PSY and one or both of the plant carotene desaturases —phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS)— as well as the cis-trans isomerases, namely ζ-carotene cis-trans Isomerase (Z-ISO; Chen et al., 2010) and carotene cis-trans isomerase (CRTISO; Isaacson et al., 2002; Park et al., 2002; Yu et al., 2011) are not. Synthesis of lycopene by PSY and CRTI in transgenic plants provides the substrate for these downstream enzymes and consequently enables the formation of the observed products.

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    Figure 2: Expression vectors used for biosynthesis of β-carotene in this study.

  • Abscisic Acid Biosynthesis, Beta-carotene => Abscisic …

    Comparison of pRSET-A and pET22b(+) for biosynthesis of β-carotene in engineered E. coli

  • Biosynthesis of Carotene in Phycomyces - [PDF …

    Figure 3: Comparison of pRSET-A and pET22b(+) for biosynthesis of β-carotene in recombinant E. coli BL21(DE3).

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Beta carotene production from industrial waste by Phycomyces nitens

In the case of sweet potatoes, breeders utilise the fact that varieties producing and storing high levels of beta-carotene (=provitamin A) are available in the Andean region of South America and thus can use these for breeding purposes and create new orange-fleshed varieties acceptable to regional taste preferences in Africa. Unfortunately, such genetic variability is not available for every crop, thus requiring the use of laternative approaches to generate the new, desirable trait.

Metabolic engineering of carotenoid biosynthesis in …

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 [7]. 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.

Metabolic engineering of carotenoid biosynthesis in plants

The primary sequence of CRTI is unrelated to the plant-type desaturases. Its structure has been partially resolved and the reaction mechanism investigated (Schaub et al., 2012). Clearly, CRTI is simpler than plant-type desaturases. CRTI employs molecular oxygen directly as an electron acceptor, while the plant enzyme utilizes plastoquinone for this purpose, and is thus linked to and dependent on complex redox-chains (Beyer et al., 1989; Mayer et al., 1990; Nievelstein et al., 1995). This electron path is also indirectly linked to molecular oxygen as the terminal electron acceptor via an oxidase identified through the immutans mutation of Arabidopsis (for review, see Kuntz, 2004). This redox pathway is especially important in non-green carotenoid-bearing tissues, while the photosynthetic electron transport is thought to play an analogous role in chloroplasts. Moreover, CRTI does not form poly-cis-configured intermediates, as plant desaturases do (Bartley et al., 1999), and therefore, cis-trans isomerases (cf Fig. 2) are not required. CRTI is also capable of introducing all four double bonds in one step.

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