A C35 carotenoid biosynthetic pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.     

Construction of carotenoid biosynthetic pathways using squalene synthase

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C₃₀ carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C₃₀ carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C₃₀ and C₄₀ carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Carbon sources-dependent carotenoid production in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effects of three phosphoenolpyruvate (PEP)-dependent PTS carbon sources (glucose, mannose and maltose) and three non-PTS carbon sources (glycerol, galactose, and lactose) on the formation of four carotenoids with diverse structures and on the cell growth of the recombinant Escherichia coli were investigated. The biosynthetic pathways of four carotenoids, C₃₀ diapolycopene, C₃₀ diapotorulene, C₄₀ lycopene, and C₄₀ beta-carotene, were engineered in E. coli. The resulting E. coli cells were grown in a mineral medium supplemented with each of the six carbon sources. Among the six carbon sources, non-PTS glycerol showed the highest performance in production of all four carotenoid structures, whereas PTS glucose showed the lowest performance. Based on the conversion yield, carotenoid-producing capability, and the cell density, we found that there was no close correlation between PTS and non-PTS transport mechanism and carotenoid formations in E. coli.     

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C₃₀ carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a “dehydrosqualene synthase.” All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.     

Redesign,Reconstruction, and Directed Extension of the Brevibacterium linens C40 Carotenoid Pathway in Escherichia coli

In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY₂ and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed β-carotene pathway synthesized an unexpected 7,8-dihydro-β-carotene in addition to β-carotene. Extension of the β-carotene pathway with the B. linens β-ring desaturase CrtU and Pantoea β-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.Carotenoids, which are produced by many microorganisms and plants, belong to a class of pigment chemicals found in nature. These structurally diverse pigments have different biological functions such as coloration, photo protection, light-harvesting, and precursors for many hormones (3, 22). Carotenoids are commercially used as food colorants, animal feed supplements and, more recently, as nutraceuticals and as cosmetic and pharmaceutical compounds (19). Currently, only a few carotenoids can be produced commercially by chemical synthesis, fermentation, or isolation from a few abundant natural sources (13). The increasing industrial importance of carotenoids has led to renewed efforts to develop bioprocesses for large-scale production of a range of carotenoids, including lycopene, β-carotene, and more structurally diverse carotenoids (17, 21, 30, 31, 34). Interestingly, a recent study showed that carotenoids with more diverse structures tend to have higher biological activity than simple structures (1).Previously, in vitro evolution altered the catalytic functions of the carotenoid enzymes phytoene desaturase CrtI and lycopene cyclase CrtY (Fig. ​(Fig.1)1) and produced novel carotenoid structures of tetradehydrolycopene and torulene in Escherichia coli (27). Furthermore, these in vitro evolved pathways and redesigned C₃₀ carotenoid biosynthetic pathways were successfully extended with additional, wild-type carotenoid modifying enzymes and evolved enzymes (21), generating novel carotenoid structures (26).Open in a separate windowFIG. 1.Reconstructed and redesigned B. linens carotenoid biosynthetic pathway in the heterologous host E. coli. Carotenogenic enzymes of B. linens, P. ananatis, and R. capsulatus, which were used for the biosynthetic pathway reconstruction, are indicated by boldface letters. Idi (IPP isomerase), IspA (FPP synthase), CrtE (GGPP synthase), CrtB (phytoene synthase), CrtI (phytoene desaturase), CrtYcYd (lycopene cyclase), CrtU (β-carotene desaturase), CrtZ (β-carotene hydrolase), CrtY₂ (mutant lycopene cyclase), and CrtA (spheroidene monooxygenase). B. linens 3,3′-dihydroxyisorenieratene biosynthesis is indicated by dashed arrows.Beside in vitro evolution (23, 34), combinatorial biosynthesis with carotenoid-modifying enzymes in a heterologous host has often been used to generate structurally novel carotenoids (24, 32). This combinatorial biosynthetic approach basically relies on the functional coordination of pathway enzymes from different sources in a heterologous host (5, 19, 35). Carotenogenic enzymes tend to be promiscuous in their substrate specificity (33) and show unexpected/hidden activities (20) when expressed in heterologous host microorganisms. One example is the unusual activity of diapophytoene desaturase CrtN in E. coli, which resulted in structurally novel compounds (20). Therefore, utilizing the promiscuity of carotenogenic enzymes makes combinatorial biosynthesis one of the most powerful strategies to generate structurally novel carotenoids that cannot be accessed in nature.Yellow colored Brevibacterium linens is commonly used as a food colorant by the cheese industry (15). Interestingly, B. linens is known to synthesize aromatic ring-containing carotenoids, isorenieratene and its hydroxy derivatives (6, 7, 16). They are produced by seven carotenogenic enzymes expressed in B. linens: GGPP synthase CrtE, phytoene synthase CrtE, phytoene desaturase CrtI, lycopene cyclase CrtYcYd, β-carotene desaturase CrtU, and the cytochrome P450 (Fig. ​(Fig.1).1). Even though the carotenoid biosynthetic pathways of B. linens have been recently studied (6, 10), there have been no systematic functional study of downstream enzymes such as lycopene cyclase CrtYcYd in the biosynthetic pathway of B. linens in a heterologous environment.Therefore, in the present study, for the first time we reconstructed, redesigned, and rationally extended the B. linens carotenoids biosynthetic pathway in E. coli to investigate the flexibility of the pathway enzymes in a heterologous host. Using this approach, we obtained an unexpected structure 3,4-didehydrolycopene, 7,8-dihydro-β-carotene, torulene, and the asymmetric carotenoid, agelaxanthin A, from engineered B. linens carotenoid pathways in E. coli.     

Novel Carotenoid Oxidase Involved in Biosynthesis of 4,4′-Diapolycopene Dialdehyde

Biosynthesis of C₃₀ carotenoids is relatively restricted in nature but has been described in Staphylococcus and in methylotrophic bacteria. We report here identification of a novel gene (crtNb) involved in conversion of 4,4′-diapolycopene to 4,4′-diapolycopene aldehyde. An aldehyde dehydrogenase gene (ald) responsible for the subsequent oxidation of 4,4′-diapolycopene aldehyde to 4,4′-diapolycopene acid was also identified in Methylomonas. CrtNb has significant sequence homology with diapophytoene desaturases (CrtN). However, data from knockout of crtNb and expression of crtNb in Escherichia coli indicated that CrtNb is not a desaturase but rather a novel carotenoid oxidase catalyzing oxidation of the terminal methyl group(s) of 4,4′-diaponeurosporene and 4,4′-diapolycopene to the corresponding terminal aldehyde. It has moderate to low activity on neurosporene and lycopene and no activity on β-carotene or ζ-carotene. Using a combination of C₃₀ carotenoid synthesis genes from Staphylococcus and Methylomonas, 4,4′-diapolycopene dialdehyde was produced in E. coli as the predominant carotenoid. This C₃₀ dialdehyde is a dark-reddish purple pigment that may have potential uses in foods and cosmetics.     

Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C₅₀ carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C₄₀ lycopene, C₄₅ nonaflavuxanthin, C₅₀ flavuxanthin, and C₅₀ sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C₅₀ carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C₅₀ carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C₅₀ carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C₅₀ carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C₅₀ carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.Carotenoids are natural pigments synthesized by bacteria, fungi, algae, and plants, and more than 750 different carotenoids have been isolated from natural sources (17). They possess important biological functions as protectants against light and oxygen excess in photosynthetic processes (32, 38), and they have been proposed to reduce the risk of certain cancers, cardiovascular disease, and Alzheimer disease due to their antioxidative properties (20, 46). The global market for carotenoids used as food colorants and nutritional supplements was estimated at approximately $935 million in 2005 (11). More than 95% of all natural carotenoids are based on a symmetric C₄₀ phytoene backbone, and only a small number of C₃₀ and even fewer C₅₀ carotenoids have been discovered (42).C₅₀ carotenoids have multiple conjugated double bonds, and they contain at least one hydroxyl group; both these features contribute to strong antioxidative properties (17, 30, 32, 38). In nature, C₅₀ carotenoids are synthesized by bacteria of the order Actinomycetales, and to date, only two different C₅₀ carotenoid biosynthetic pathways have been described in the literature. The biosynthetic pathways of the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-ɛ,ɛ-carotene] and the β-cyclic C₅₀ carotenoid C.p.450 [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-β,β-carotene] have been elucidated in Corynebacterium glutamicum (22, 23) and in Dietzia sp. CQ4 (41), respectively. For both pathways, the common precursor, C₄₀ lycopene, is synthesized from C₁₅ farnesyl pyrophosphate (FPP) via the methylerythritol 4-phosphate (MEP) pathway, which is present in most eubacteria (33). Effective lycopene production has been achieved in genetically engineered noncarotenogenic hosts, such as Escherichia coli and Saccharomyces cerevisiae (9). Accordingly, the potential of using such biotechnologically relevant hosts for heterologous production of any lycopene-derived carotenoids has generated high interest.The biosynthesis of cyclic C₅₀ carotenoids from lycopene is catalyzed by lycopene elongase and carotenoid cyclases. Even though most carotenoids in plants and microorganisms exhibit cyclic structures, cyclization reactions were predominantly known for C₄₀ pathways (45) catalyzed by monomeric enzymes that have been isolated from plants and bacteria (5, 16, 27, 29, 31, 36). In C. glutamicum, the genes crtYe, crtYf, and crtEb were identified as being involved in the conversion of lycopene to the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin (22, 44). Sequential elongation of lycopene into the acyclic C₅₀ carotenoid flavuxanthin was catalyzed by the crtEb gene product lycopene elongase. Subsequent cyclization to decaprenoxanthin was catalyzed by a heterodimeric C₅₀ carotenoid, ɛ-cyclase, encoded by crtYe and crtYf (22). C. glutamicum can synthesize both mono- and diglucosylated decaprenoxanthin; however, the genetic and enzymatic bases for glucosylation of decaprenoxanthin are unknown. Analogous to decaprenoxanthin, biosynthesis of the β-cyclic C₅₀ carotenoid C.p.450 in Dietzia sp. CQ4 from lycopene involves lycopene elongase and C₅₀ carotenoid β-cyclase activities (41).While most cyclic carotenoids exhibit β-rings, ɛ-ring-containing pigments are common in higher plants (7), and carotenoids substituted only with γ-rings are rarely observed in plants and algae (14). To date, no biosynthetic pathway for γ-cyclic C₅₀ carotenoids has been reported in the literature.Micrococcus luteus NCTC2665 (the “Fleming strain”) is a Gram-positive bacterium belonging to the family Micrococcaceae within the order Actinomycetales. The carotenoids, including the γ-cyclic C₅₀ sarcinaxanthin [(2R,6R,2′R,6′R)-(2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-γ,γ-carotene)], synthesized by this bacterium have been identified and structurally elucidated (26). We recently isolated and characterized several wild-type M. luteus strains from the sea surface microlayer of the middle part of the Norwegian coast (39). Here, we report one additional such marine M. luteus isolate, designated Otnes7, forming color-intensive colonies indicating high sarcinaxanthin production levels. Both Otnes7 and NCTC2665 were used as M. luteus model strains, and the sarcinaxanthin biosynthetic gene clusters were cloned from both strains. The complete sarcinaxanthin biosynthetic pathway from lycopene was elucidated, including glucosylation, and we also explored the potential of using Otnes7-derived genes to achieve effective heterologous production of sarcinaxanthin in E. coli. The results add important new knowledge of the biosynthesis of C₅₀ carotenoids, and in particular, they highlight the diverse functions of C₅₀ carotenoid cyclases leading to synthesis of structurally different carotenoids.     

Evolution of a pathway to novel long-chain carotenoids

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.     

Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C(30), C(35), C(40), C(45), C(50) carotenoids

At least 700 natural carotenoids have been characterized; they can be classified into C(30), C(40) and C(50) subfamilies. The first step of C(40) pathway is the combination of two molecules of geranylgeranyl pyrophosphate to synthesize phytoene by phytoene synthase (CrtB or PSY). Most natural carotenoids originate from different types and levels of desaturation by phytoene desaturase (CrtI or PDS+ZDS), cyclization by lycopene cyclase (CrtY or LYC) and other modifications by different modifying enzyme (CrtA, CrtU, CrtZ or BCH, CrtX, CrtO, etc.) of this C(40) backbone. The first step of C(30) pathway is the combination of two molecules of FDP to synthesize diapophytoene by diapophytoene synthase (CrtM). But natural C(30) pathway only goes through a few steps of desaturation to form diaponeurosporene by diapophytoene desaturase (CrtN). Natural C(50) carotenoid decaprenoxanthin is synthesized starting from the C(40) carotenoid lycopene by the addition of 2 C(5) units. Concerned the importance of carotenoids, more and more attention has been concentrated on achieving novel carotenoids. The method being used successfully is to construct carotenoids biosynthesis pathways by metabolic engineering. The strategy of metabolic engineering is to engineer a small number of stringent upstream enzymes (CrtB, CrtI, CrtY, CrtM, or CrtN), then use a lot of promiscuous downstream enzymes to obtain large number of novel carotenoids. Two key enzymes phytoene desaturase (CrtI(m)) and lycopene cyclase (CrtY(m)) have been modified and used with a series of downstream modifying enzymes with broad substrate specificity, such as monooxygenase (CrtA), carotene desaturase (CrtU), carotene hydroxylase (CrtZ), zeaxanthin glycosylase (CrtX) and carotene ketolase (CrtO) to extend successfully natural C(30) and C(40) pathways in E. coli. Existing C(30) synthase CrtM to synthesize carotenoids with different chain length have been engineered and a series of novel carotenoids have been achieved using downstream modifying enzymes. C(35) carotenoid biosynthesis pathway has been constructed in E. coli as described. C(45) and C(50) carotenoid biosynthesis pathways have also been constructed in E. coli, but it is still necessary to extend these two pathways. Those novel acyclic or cyclic carotenoids have a potential ability to protect against photooxidation and radical-mediated peroxidation reactions which makes them interesting pharmaceutical candidates.     

Functional Characterization of the Xanthophyllomyces dendrorhous Farnesyl Pyrophosphate Synthase and Geranylgeranyl Pyrophosphate Synthase Encoding Genes That Are Involved in the Synthesis of Isoprenoid Precursors

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C₂₀) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C₅) and dimethylallyl pyrophosphate (DMAPP, C₅) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C₁₅) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C₁₀) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.     

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

Background

Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data.

Methodology/Principal Findings

Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection.

Conclusions/Significance

Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a “bramble” model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic “root”. Structural diversification may be constrained (“trimmed”) where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification.     

Substrate specificity of acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacterium producing myristoleic acid

Chlorophyll b-containing cyanobacterium Prochlorothrix hollandica is characterized by a high content of esterified fatty acids (FA) with 14 and 16 carbon atoms in the membrane lipids. Depending on the conditions of cultivation, the relative amount of myristic (C_14:0) and myristoleic (C_14:1) acids can reach 35%, and palmitic (С_16:0) and palmitoleic (С_16:1) acids can reach 60% of the sum of all fatty acids in cells. Monounsaturated FAs are represented by C_14:1, and C_16:1 with an olefinic bond presumably located in the Δ⁹ position. We cloned the gene of acyl-lipid Δ9-desaturase, desC1, from Prochlorothrix hollandica and characterized its specificity to the length of the substrate using the heterologous expression in Escherichia coli cells adding C_14:0 or stearic (C_18:0) acids as exogenous substrates. The results show that DesC1 Δ⁹ desaturase generates olefinic bonds in the FAs with a length of 14 to 18 carbon atoms with an approximately equal efficiency. This indicates that the length of the FA chain in P. hollandica is determined by the activity of the FA synthase, and the chain is desaturated at the Δ⁹ position nonspecifically relatively to its length.     

Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C₃ to C₅, medium-chain-length (MCL) PHAs consist of monomer units of C₆ to C₁₄, and SCL-MCL PHAs consist of monomers ranging in size from C₄ to C₁₄. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C₄ to C₁₀ and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.     

RNA interference-mediated repression of MtCCD1 in mycorrhizal roots of Medicago truncatula causes accumulation of C27 apocarotenoids, shedding light on the functional role of CCD1

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C₄₀ carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C₁₃ cyclohexenone and C₁₄ mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C₂₇ apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C₂₇ apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C₂₇ apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C₂₇ → C₁₄ + C₁₃), while the first step (C₄₀ → C₂₇ + C₁₃) may be catalyzed by CCD7 and/or CCD4 inside plastids.     

Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.     

Cloning,sequencing and expressing the carotenoid biosynthesis genes,lycopene cyclase and phytoene desaturase,from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).     

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.     

Bleaching herbicide flurtamone interferes with phytoene desaturase

The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of α- and β-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I₅₀ value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K₁ value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport.