Recombinant Squalene Synthase. Synthesis of Non-Head-to-Tail Isoprenoids in the Absence of NADPH

Jarstfer, Michael B.; Zhang, Dong Lu; Poulter, C. Dale

doi:10.1021/ja020410i

Cited by 52 publications

(73 citation statements)

References 61 publications

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“…FPP is first converted to the intermediate PSPP, followed by its reductive rearrangement to squalene (24). However, PSPP is not evident in these reactions unless NADPH, the reducing reagent, is omitted from the incubations (19). Under conditions of adequate NADPH, it appears unlikely that PSPP is released from the squalene synthase enzyme, then rebound as a natural consequence of the catalytic cycle (34).…”

Section: Discussionmentioning

confidence: 99%

“…Our approach for identifying the triterpene biosynthetic genes in B. braunii has relied in large part on the putative similarities in the biosynthetic mechanisms for squalene and botryococcene (19)(20)(21). Squalene biosynthesis has been extensively investigated because it is positioned at a putative branch point in the isoprenoid biosynthetic pathway directing carbon flux to sterol metabolism, and thus represents a potential control point for cholesterol biosynthesis in man (22).…”

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confidence: 99%

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Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Niehaus

Okada

Devarenne

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

138

153

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Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.algae | biofuels | terpene enzymology

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Niehaus

Okada

Devarenne

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

138

153

View full text Add to dashboard Cite

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“…Unable to convert into phytoene in wild-type CrtM, prephytoene diphosphate either remains stuck in the enzyme, departs, or undergoes other types of rearrangement to yield noncarotenoid products. (A similar phenomenon is well known for SqS; in the absence of NADPH, which is required to convert presqualene diphosphate to squalene, SqS produces a complex mixture of nonsqualene compounds including rillingol, 10-hydroxybotryococcene, and 12-hydroxysqualene [22,89].) However, in CrtM mutants where F26 or W38 is replaced with a smaller or more flexible amino acid, the prephytoene diphosphate formed from two molecules of C 20 PP is efficiently rearranged to form phytoene.…”

mentioning

confidence: 55%

“…The biosynthesis of carotenoid backbones (and squalene) has proven to be a complex process (22,88,89) (Fig. 5a).…”

Section: Directed Evolution Of Key Carotenoid Biosynthetic Enzymesmentioning

confidence: 99%

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Umeno

Tobias

Arnold

2005

Microbiol Mol Biol Rev

190

148

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Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed

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“…This mechanism is virtually identical to that of squalene synthase (SqS), the enzyme that catalyzes the first step in cholesterol biosynthesis. Indeed, when deprived of NADPH, SqS produces product 1 as the main product (3,11). Carotene synthases are similar to SqS in sequence and predicted secondary structure; they probably share a common ancestor and have virtually identical folds.…”

Section: And C 30 Carotenoid Synthase Activities Of Crtm Variantsmentioning

confidence: 99%

Evolution of a Pathway to Novel Long-Chain Carotenoids

Umeno

Arnold

2004

J Bacteriol

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Using methods of laboratory evolution to force the C 30 carotenoid synthase CrtM to function as a C 40 synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C 45 and C 50 carotenoid backbones (mono-and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.Carotenoids are natural pigments with important biological activities (4,10,16,17). Most are based on a 40-carbon (C 40 ) phytoene backbone produced by condensation of 2 molecules of geranylgeranyldiphosphate (GGDP; C 20 PP), a reaction catalyzed by the carotenoid synthase CrtB (Fig. 1). The vast majority of the Ͼ700 known carotenoids (9) arise as a result of different types and levels of modification of the C 40 backbone, catalyzed by promiscuous (downstream) carotenoid biosynthetic enzymes (5). A few bacteria, notably Staphylococcus and Heliobacterium spp. (23,24), have a C 30 pathway, which starts with the CrtM synthase-catalyzed condensation of 2 molecules of farnesyldiphosphate (FDP; C 15 PP) to form 4,4Ј-diapophytoene. Yet other bacteria (such as Corynebacterium and Halobacterium spp.) are known to accumulate C 50 carotenoids, but these longer-chain structures are biosynthesized starting from the C 40 structure by the addition of 2 C 5 (isoprene) units (14). Various longer isoprenyldiphosphates are made by different organisms (30) and are potential precursors for longerchain carotenoids. They are precursors to other biosynthetic pathways, however, and no known carotenoids are derived from them. Thus, carotenoid size is tightly controlled by the carotene synthase reaction (20,27).To create new pathways for the biosynthesis of carotenoids with backbones larger than C 40 , we focused on engineering the carotenoid synthase to accept longer diphosphate substrates. Very little is known of the structure or basis for the specificity of these membrane-associated enzymes. Using random mutagenesis and a functional complementation screen for C 40 synthase activity, however, we identified single-amino-acid substitutions in the C 30 synthase CrtM (F26L or F26S) that confer the C 40 function (27). By repeating this experiment with a random mutant library that was free from mutation at F26, we recently found two more amino acid substitutions, W38C and E180G, that confer the same phenotype (26). Upon further mutagenesis at these three residues, we show here that the specificity of the carotenoid synthase CrtM is controlled at the second (rearrangement) step of its two-step reaction. Furthermore, we have engineered synthase variants that can make ...

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Recombinant Squalene Synthase. Synthesis of Non-Head-to-Tail Isoprenoids in the Absence of NADPH

Cited by 52 publications

References 61 publications

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Evolution of a Pathway to Novel Long-Chain Carotenoids

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