- freely available
Mar. Drugs 2014, 12(9), 4810-4832; doi:10.3390/md12094810
Abstract: Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.
Carotenoids are a class of naturally occurring pigments originated in the chloroplasts and chromoplasts of plants, algae and some photosynthetic microorganisms [1,2,3,4]. As of 2004, over 750 known carotenoids, which can be divided into xanthophylls (containing oxygen) and carotenes (pure hydrocarbons), have been isolated from natural sources . These structurally diverse pigments play important biological roles in light capture, protection of cells from the damaging effects of free radicals, and synthesis of many hormones as a precursor [6,7,8,9,10]. Carotenoids are traditionally used as food colorants, animal feed supplements, and, very recently, as nutraceuticals and pharmaceuticals [11,12]. Over the past few decades, researches have supported that the ability of carotenoids to reduce the risk of certain cancers, cardiovascular diseases, and degenerative pathogenesis (e.g., Alzheimer and Parkinson) due to their antioxidative properties [13,14]. According to “Carotenoids: A Global Strategic Business Report” from Global Industry Analysts (GIA), the global market for carotenoids was estimated at approximately $1.07 billion in 2010 and is projected to top $1.2 billion by 2015 . Therefore, many efforts have been made to improve the production of these natural compounds for ever-increasing demands [12,16,17].
The ocean is a complex aquatic ecosystem covering about 71% of the Earth’s surface, which is around 300 times larger than the habitable volume of the terrestrial habitats on Earth. A large proportion of all life on Earth lives in the ocean. Ecologically distinct from the terrestrial ecosystem, the ocean constitutes a unique reservoir of marine biodiversity and provides a vast resource of foodstuffs, medicines, and other useful materials. As such, more than 250 novel carotenoids have originated from marine species , many of which show great potential in commercial applications . With the advent of synthetic biology and metabolic engineering, many engineering tools including vectors, genetic controllers, and enzyme designing, have been developed for heterologous production of valuable chemicals. These tools create new opportunities for exploring marine carotenoids for food and health industries. In this review, we describe diverse and novel carotenoids from marine resources and summarize recent progresses in synthetic biology and metabolic engineering which provide great application potential for marine carotenoids.
2. Diversity of Marine Carotenoids
Many carotenoids have been reported from a wide range of marine species. The advances in current technologies facilitate the elucidation of the carotenoid biosynthetic pathways and relevant enzymes from marine species, which would enable the production of important carotenoids from marine organisms.
2.1. Pathways and Diverse Enzymes for Biosynthesis of Carotenoids
Biosynthetic routes to carotenoids begin with the basic building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), although carotenoids are very diverse in chemical structure. Two distinct pathways, the 2-C-methyl-d-erythritol 4-phospahte (MEP) pathway and the mevalonic acid (MVA) pathway, are responsible for the synthesis of IPP and DMAPP. These two pathways have been reviewed in detail elsewhere [19,20,21,22]. IPP and DMAPP are head-to-tail condensed to generate farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) by isoprenyl diphosphate synthases (e.g., IspA of Escherichia coli and CrtE of Pantoea agglomerans) [23,24]. As shown in Figure 1, FPP and GGPP are further head-to-head condensed to produce symmetric hydrosqualene (C30) and phytoene (C40), which are dehydrogenated in a stepwise manner by desaturating enzymes representing an important branch point for pathway diversification [25,26].
Enzymes involved in the biosynthesis of carotenoids have been mainly investigated in carotenogenic cyanobacteria and land plants [27,28]. They are mostly associated with cytoplasmic and organelle membranes where the hydrophobic substrates of carotenogenic enzymes are located . So far, very few crystal structures of carotenogenic enzymes have been elucidated because of their association with the membranes [30,31]. More than 95% of carotenoids have been characterized in nature to be phytoene-based , which will be extensively discussed in this review.
Phytoene synthase is positioned early in the carotenoid synthesis pathway and is responsible as a pathway gatekeeper to discriminate GGPP substrate from enormous isoprenyl diphosphates . Phylogenic analysis of 20 phytoene synthases from marine organisms supports the endosymbiotic theory that plastids evolve from a cyanobacterium, which is engulfed and retained by a unicellular protist [33,34]. Cyanobacteria Acaryochloris marina and Prochlorococcus marinus are clustered with green algae and land plant tomato (Figure 2A). However, phytoene synthases still display a significant diversification by evolution. A consensus position of 24.5% (identity of 0.5%) is remained among phytoene synthases from marine algae, bacteria, Achaea and land plants. There is only a similarity of 31.9% even between the two proteobacteria phyla α-proteobacteria and γ-proteobacteria.
The photochemical properties of a carotenoid depend on the size of the chromophore formed by conjugated double bonds, and a C40 backbone can accumulate up to 15 conjugated double bonds . Thus, six sequential desaturation steps are required to dehydrogenate colorless phytoene, which has three conjugated double bonds in the center . Lycopene containing a chromophore with eleven conjugated double bonds is the direct precursor of α/β/γ-carotenes or isorenieratene, the phytoene-based C40 carotenoid backbone (Figure 1). In general, oxygenic phototrophs require three enzymes, phytoene desaturase, ζ-carotene desaturase and cis-carotene isomerase to generate lycopene . However, most bacterial phytoene desaturases are able to catalyze all three reactions . There are also some organisms that disobey this general rule. Primitive cyanobacteirum Gloeobacter violaceus PCC 7421 uses bacterial type phytoene desaturase, and no homolog of ζ-carotene desaturase or cis-carotene isomerase is found in its genome [36,37]. Among anoxygenic phototrophs, green sulfur bacteria use three enzymes to catalyze desaturation, whereas purple bacteria, green filamentous bacteria, and heliobacteria use only one enzyme [38,39]. Phytoene desaturases also exhibit significant diversities among different organisms (Figure 2B). Just as phytoene synthase, green algae are clustered with tomato but they are distinguished from cyanobacteria. There is just a similarity of 23.2% among 21 proteins. It is suggested that phytoene desaturase exhibits a much faster evolution from the ancestral blueprint and higher diversities among species than phytoene synthases, which may correspond to promiscuous activities of phytoene desaturase.
2.2. Diversity of Marine Carotenoids
Carotenogenic organisms in ocean are algae and bacteria, which possess all the genes for de novo synthesis of carotenoids [2,3,4]. Unicellular microalge Dunaliella salina and Dunaliella bardawil are rich in the orange pigment β-carotene (HaH'a, Figure 1) [41,42]. Two rings of β-carotene are often oxidized to form astaxanthin (Ha1H'a1, Figure 3) in some microalgae by β-carotene hydroxylase and ketolase , which can individually catalyze the modification of β-carotene to generate zeaxanthin (Ha2H'a2, Figure 3) in Spriulina platensis and Spriulina maxima , and canthaxanthin (Ha3H'a3, Figure 3) in Haematococcus pluvialis, Clorella vulgaris and Colastrella striolata [44,45,46]. The modifications can just occur in one ring to generate asymmetric intermediates such as β-cryptoxanthin (HaH'a2, Figure 3) and echinenone (HaH'a3, Figure 3). Chlorophyta Scenedesmus almeriensis and Muriellopsis sp. accumulate a large amount of lutein (Hb1H'a2, Figure 3), which is derived from α-carotene (HbH'a, Figure 1) . Cryptophyta also synthesize α-carotene as well as acetylenic derivatives crocoxanthin (HbH'a4, Figure 3) and monadoxanthin (Hb1H'a4, Figure 3) . Acetylenic groups are also found in β-carotene derivatives alloxanthin (Ha4H'a4, Figure 3) in Cryptophyta , and diatoxanthin (Ha2H'a4, Figure 3) and epoxy oxidized diadinoxanthin (Ha4H'a5, Figure 3) in Heterokontophyta, Haptophyta, Dinophyta, and Euglenophyta [28,49,50]. The unique acetylenic carotenoids are only found in algae. In brown algae and diatoms, acetylated and unique allenic modifications produce dinoxanthin (H'a5Ha6, Figure 3) and chain-oxidized fucoxanthin (Ha6H'a7, Figure 3) [2,51]. Some Chlorophyta species modify the methyl group of lutein to generate loroxanthin (Hb1H'a8, Figure 3) in Scenedesmus obliquus and Chlorella vulgaris , and siphonaxanthin (Hb1H'a9, Figure 3) in Codium fragile . Aromatic isorenieratene (HcH'c, Figure 1) is a usual biomarker compound, which is synthesized from β-carotene in actinobacteria or γ-carotene (H'oHa, Figure 1) in green and purple sulfur bacteria [54,55]. γ-Carotene can also be converted to chlorobactene (HcH'o, Figure 1) and OH-chlorobactene (HcH'o1, Figure 3). Glycoside modifications generate OH-chlorobactene glucoside (HcH'o2, Figure 3) in green sulfur bacteria and myxol 2'-fucoside (Ho3H'a2, Figure 3) in Cyanophyta [54,56]. Dinophyta can synthesize C37-skeletal carotenoids such as peridinin (Hd1H'a6, Figure 3) . Animals do not have pathways for de novo synthesis of carotenoids, but they obtain carotenoids from food and further modify carotenoids by oxidation, reduction, translocation of double bonds, cleavage of double bonds, etc. Peridinin-originated carotenoids such as peridininol (He1H'd1, Figure 3) and cyclopyrrhoxanthin (He2H'd2, Figure 3) have been isolated from bivalves Crassostrea gigas, Paphia amabillis, and Corbicula japonica [58,59,60]. Two unique nor-carotenoids, 2-nor-astaxanthin (Hf1H'a1, Figure 3) and actinoerythrin (Hf2H'f2, Figure 3), have been found in sea anemones Actinia equine and Tealia feline . The carotenoid diversity in marine animals has been well summarized in detail elsewhere . It is also worthy to note that some carotenoids are present in different stereo configurations among organisms (not covered in this review), which also greatly contributes to the diversification of carotenoids.
2.3. Synthesis of Some Important Marine Carotenoids and Enzymes
β-Carotene as well as xanthophylls astaxanthin, zeaxanthin, lutein, and fucoxanthin are some representative marine carotenoids due to their abundance in marine organisms and their inherent antioxidant properties. β-Carotene is synthesized from the cyclization of lycopene, a key step in generating carotenoid diversity by lycopene cyclases, which can also lead to α/γ-carotene formation (Figure 1). The β-cyclase catalyzes the symmetrical formation of two identical β-ionone rings of β-carotene. On the other hand, α-carotene contains two different ring structures (ε and β) formed by the action of additional ε-cyclase with β-cyclase. Four distinct families of lycopene cyclases, CrtY-type β-cyclases in proteobacteria, CrtL β/ε-cyclases in some cyanobacteria, the heterodimeric cyclases in some Gram-positive bacteria and FixC dehydrogenase superfamily lycopene cyclases in Chlorobium tepidum and Synechococcus sp. PCC 7002, have been identified to date . Further decorations occur via a variety of ketolation (oxidation), hydroxylation (Figure 4), which are the major causes for the diversity among carotenoids . β-Carotene ketolase (CrtW or CrtO) adds the keto groups at the 4,4′-position of the ring and β-carotene hydroxylase (CrtZ) adds the hydroxyl group at the 3,3′-position . Both enzymes are responsible for the formation of astaxanthin via zeaxanthin or canthaxanthin routes in some cyanobacteria and algae (Figure 3). Lutein formation is ascribed to the hydroxylation of α-carotene by cytochrome P450 enzymes in Arabidopsis thaliana , while the pathway and enzymes remain to be elucidated from marine organisms. Fucoxanthin with a unique allenic and epoxide structure is derived from zeaxanthin in brown seaweeds, diatoms and dinoflagellates. Genome analysis indicates that zeaxanthin epoxidases epoxidize zeaxanthin to form antheraxanthin (Ha2H'a5, Figure 3) and violaxanthin (Ha5H'a5, Figure 3) . Two possible routes have been proposed for the synthesis of fucoxanthin from violaxanthin via neoxanthin (He1H'a5, Figure 3) or diadinoxanthin . Very recently, a cytochrome P450-type carotene hydroxylase (PuCHY1) has been isolated from red alga Porphyra umbilicalis. The compensatory expression of PuCHY1 results in the formation of violaxanthin, neoxanthin, and lutein in A. thaliana by the β/ε-hydroxylation activities . Some of the carotenogenic enzymes characterized from marine organisms have been summarized in the literature .
3. Technology Developments for Production of Carotenoids
Over the decades, many researches have been done for the production of carotenoids. Carotenogenic pathways have been identified and manipulated in several organisms, and advances in metabolic engineering and synthetic biology have resulted in significant improved production of carotenoids including astaxanthin, zeaxanthin, and lutein.
3.1. Easy Colorimetric Screening of Production of Carotenoids
Carotenoids contain chromophores absorbing visible light and appear as being yellow (e.g., β-carotene) to red (e.g., lycopene), which benefits carotenogenic gene mining and engineering upon carotenoid synthesis pathway. To date, many carotenoid biosynthetic genes have been cloned from plants, bacteria, and fungi based on their abilities to render different colors to the host [68,69,70]. This merit has been vigorously implemented for random mutagenesis, directed evolution, and proof-of-principle experiments in synthetic biology. Moreover, cellular carotenoids can be easily extracted into an organic solvent and differentiated in a sensitivity of submilligrams per liter with a linear correlation between carotenoid contents and color intensity [71,72]. This provides an easy and high-throughput way to evaluate the performance of newly built synthetic circuits or methodologies for improved biosynthesis of carotenoid (Figure 5A).
3.2. Pathway Engineering for Production of Carotenoids
Carotenoid biosynthesis emerges from the central isoprenoid pathway, either the MEP pathway or the MVA pathway, existing in all organisms [19,22]. The expression of carotenogenic genes can yield carotenoids of interest in a heterologous organism [16,73,74,75]. The early attempts led to the production of lycopene, β-carotene, and astaxanthin in Saccharomyces cerevisiae and Candida utilis by the expression of carotenogenic enzymes from Pantoea ananatis [74,76]. Corynebacterium glutamicum is a native producer of decaprenoxanthin and its glucosides, and it has been engineered to synthesize C50 carotenoids C.P.450 and sarcinaxanthin . To date, there have been many exemplary illuminations to achieve high carotenoid titers from non-native producers. Carotenogenic enzymes from different sources exhibit different capacities in carotenoid biosynthesis. A two-fold higher lycopene production is obtained in E. coli by the expression of carotenogenic enzymes from P. agglomerans (27 mg/L) than from P. ananatis (12 mg/L) . Metabolic engineering approaches allow the assembly of genes from different organisms for production purposes or for building new carotenoids [32,79,80]. β-Carotene production has been improved by hybrid expression of carotenogenic genes from P. agglomeras and P. ananatis in E. coli . In another example, expression of β-end ketolase from Agrobacterium aurantiacum extends the zeaxanthin β-d-diglucoside pathway from P. ananatis, and synthesizes novel astaxanthin β-d-diglucoside and adonixanthin β-d-diglucoside . Generally, a sufficient precursor supply is a prerequisite for high-yield production of carotenoids. Overexpression of the rate-limiting enzymes 1-deoxy-d-xylulose-5-phosphate synthase and reductoisomerase led to a 3.6-fold increase in lycopene production in E. coli when compared with the native MEP pathway for IPP and DMAPP supply . Overexpression of the rate-limiting enzyme 3-hdroxy-3-methyl-glutaryl-coenzyme A (HNG-CoA) reductase of the MVA pathway from Xanthophyllomyces dendrorhous significantly increased β-carotene production in S. cerevisiae . A great effort in metabolic engineering of the central carotenoid building block pathway is the introduction of a hybrid MVA pathway of Streptococcus pneumonia and Enterococcus faecalis into E. coli, which enables the recombinant host to produce 465 mg/L of β-carotene . With more available genetic tools, microbial organisms such as Pseudomonas putida and Bacillus subtilis have also been developed as platform hosts for carotenoid production [84,85].
Carotenoids synthesis involves multiple enzymes . The expression level of all the components of a multigene circuit should be orchestrated to optimize metabolic flux and to gain a high yield (Figure 5B) . A random approach is screening of the best orchestra from numerous combinatorial assemblies of required genes and control elements. BioBrick™ paradigm is capable of rapidly assembling a biosynthetic pathway in a variety of gene orders from different promoters in plasmids with different copy numbers . It is possible to build a hybrid carotenoid pathway wherein each enzyme possesses a right turnover number, however, BioBrick™ assembly is still not in a high throughput to create vast combinatorial expression constructs for the best combination of carotenogenic genes. Recently, several advanced assembly methods using homologous recombination, such as sequence and ligation-independent cloning (SLIC), Gibson DNA assembly and reiterative recombination, have been applied to construct multigene circuits [88,89,90]. These advances promise to randomize all genetic components, including genes, promoters, ribosome binding sites, and other control modules to build a large number of individual genetic circuits for screening purposes. A so-called “randomized BioBrick assembly” approach has been applied to the optimization of the lycopene synthesis pathway wherein the expression construct was designed to independently express each enzyme from its own promoter, which resulted in an increase by 30% in lycopene production . A longer and more complicated pathway can be modularized into subsets, which contain pathway enzymes with similar turnover numbers. Modulating these subsets would be more convenient and efficient than regulating all components of the entire pathway for improved production . By using this multivariate modular metabolic engineering (MMME) approach, recent work achieved a 15,000-fold increase in production of taxadiene, a precursor of the anti-cancer drug taxol . There are also a variety of promising approaches, such as tunable promoters, tunable intergenic regions, and ribosome binding site design, which can be applied to fine tuning the expression of modules [94,95,96]. In the other approaches, a multi-genic operon is transcribed into a single polycistronic mRNA, and then the large transcript can be spliced to small monocistronic transcripts through post-transcriptional RNA processing such as ribozyme cleavage and clustered regularly interspaced short palindromic repeats (CRISPR) editing. Thus, the stability of the monocistronic transcripts can be independently modulated to differentiate the expression level of each enzyme even in a multi-gene operon. These RNA processing tools have been developed as insulating elements between operonic genes to reduce the context dependence of the genes in a polycistronic transcription unit . The diffusion of pathway intermediates can decrease the effective concentrations of intermediates for following enzyme reactions and some intermediates may serve for competing pathways. By learning from Mother Nature, synthetic biologists spatially organize enzymes of the MVA pathway by protein scaffolds in E. coli to minimize diffusion limitation and achieve a 77-fold increase in mevalonic acid production . The propanediol utilization machinery of Citrobacter freundii has been heterologously recasted in E. coli . Some intermediates of carotenoid synthesis such as isoprenyl diphosphates are toxic when they accumulate over the concentration threshold . To avoid the accumulation of toxic intermediates, genetic sensors can potentially be coupled with gene expression cassettes to regulate the intermediate flux in a dynamic manner. The native E. coli promoters that respond to the toxic FPP have been successfully used to dynamically regulate the amorphadiene synthesis pathway and improve the production by two-fold over common inducible promoters and constitutive promoters . The Ntr regulon has been engineered to control lycopene synthesis in response to glycolytic flux dynamics, resulting in an 18-fold increase in lycopene production .
3.3. Genome Engineering for Strain Development
For the most efficient carotenoid production, the biological system of the host organism also needs to be optimized, by, for example, redirecting cellular carbon flux to the carotenoid synthesis pathway. The de novo synthesis of carotenoids is initiated from acetyl-coA by the MVA pathway or glycolytic metabolites pyruvate and glycraldehyde-3-phosphate (G3P) by the MEP pathway. The direct efforts are focused on the modification of associated genes to these pathways. Deletion of pyruvate kinases PykFA can balance the availability of pyruvate and G3P for the MEP pathway, and increase lycopene production by 2.8-fold in E. coli . The deletion of glucose-6-phospahte (G6P) dehydrogenase Zwf, which branches G6P to pentose phosphate pathway results in an increase by 30% in lycopene production . Deletion of carbohydrate phosphotransferase system yields a seven-fold increase in lycopene production in another study . Replacement of native promoters of the rate-limiting genes of the MEP pathway with the T5 promoter has been carried out for enhancement of the targeted pathway flux, which results in a 4.5-fold increase in β-carotene production .
A heterologous pathway is not just an independent entity. It communicates with the native cellular metabolism and is therefore governed by the global regulation of the host organisms. Adaptive laboratory evolution is a traditional route for strain engineering to achieve desirable industrially relevant phenotypes. Owing to the antioxidant properties of carotenoids, adaptive evolution has been successfully applied to an engineered S. cerevisiae with periodic hydrogen peroxide shocking, resulting in a three-fold increasee of β-carotene production. Subsequent transcriptome analysis indicates that some genes related with lipid biosynthesis and MVA pathways are up-regulated in the adopted strains . It also suggests that carotenoid production can be improved by modifications (knock-out or overexpression) of distant genes, which are responsible for the overall regulation of the metabolic network or the physiological fitness of the host (Figure 5C). In a genome-wide screening of yeast deletion collection, 24 deletions exhibit significant higher carotenoid levels than the wild type. The triple deletion of ROX1, YJL064W, and YJL062W shows an almost four-fold increase in total carotenoid production . Gene deletions of hnr, yjfP, and yjiD related to the improvement of lycopene production have been identified from a global transposon E. coli mutant library . Other gene deletions such as gdhA, cyoA, ppc, gpmA, gpmB, eno, glyA, aceE, talB, and fdhF have been in silico identified using a stoichiometric model . The triple mutation of gdhA, aceE and fdhF was validated to increase lycopene production by nearly 40% in E. coli over the engineered parental strain. A similar set of gene deletions dhA, cyoA, gpmA, gpmB, icdA, and eno have been also in silico identified using different metabolic network models . Overexpression of some genes encoding global regulatory proteins AppY, Crl, RpoS, and ElbAB, oxidoreductases TorC, YdgK, and YeiA, and hypothetical proteins YedR and YhbL, result in a significant increase in lycopene production in E. coli . With a profound understanding of the landscape of genome manipulation, all these knocked-out and overexpressed alleles have been combined and optimized to generate high-fitness host strains for lycopene production [113,114]. ATP and NADPH are also important cofactors for the production of carotenoids. Using engineering ATP synthesis, pentose phosphate and TCA modules, recent work has shown the highest β-carotene production of 2.1 g/L by a fed-batch fermentation process in E. coli . The advances in synthetic biology greatly boost genome manipulation on a large scale. Multiplex automated genome engineering (MAGE) simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells by directing ssDNA to the lagging strand of the replication fork during DNA replication . The modifications can cover gene inactivation, expression regulations, and so on. Aforementioned twenty genes related to lycopene production have been targeted to tune their expression using a complex pool of synthetic DNAs, and lycopene production is increased more than five-fold. A complementary method called trackable multiplex recombineering (TRMR) has been developed to simultaneously map genome modifications that affect a trait of interest, which combines parallel DNA synthesis, recombineering and molecular barcode technology to enable rapid modification of all E. coli genes in an a priori knowledge-independent way .
Metabolic engineering for the production of valuable compounds often heavily relies on plasmid-based expression of the synthesis pathway in a heterologous host. Although plasmids are easily manipulated and allow strong expression of targeted enzymes, the plasmid-based systems suffer from genetic instability such as plasmid loss, an additional antibiotic cost, and a potential risk of antibiotic marker spreading to other organisms . Accordingly, chromosomal integration of the production pathway promises the host to achieve stable overproduction of the desirable chemicals including carotenoids. By λ-Red homologous recombination, plasmid-free engineered E. coli strain has been developed to produce lycopene and astaxanthin . The expression cassettes can be integrated into different loci to increase the number of gene copies. P1 transduction usually plays a role in transfering the different alleles between host strains. Recently, an intelligent strategy called chemically inducible chromosomal evolution (ClChE) has been developed to reduce the daunting repeated one-at-a-time tasks in the chromosomal integration of target genes . ClChE allows the host to acquire a high gene copy (up to 40 copies) expression of integrated pathways with increasing concentration of selective chemicals, and the increased copy number is stabilized by the removal of the recA gene. With this approach, lycopene production has been increased by 60% from single copy integrated strain. The ClChE strategy has been further modified to eliminate antibiotic marker for environmental safety and health issue after the evolution of the recombinant host strain .
3.4. Protein Engineering for Improvement of Carotenoid Production Enzymes
Pathway engineering for efficient production of desired chemicals is often challenged by limitations associated with the pathway enzymes themselves, such as low turnover numbers and promiscuities generating unwanted by-products . Protein engineering provides a powerful solution to improve specific activity and substrate specificity of enzymes, and even to create new activity. Methods of protein engineering include directed evolution and computer-assisted rational design (Figure 5D) [122,123]. Directed evolution is an iterative process that imitates Darwinian evolution in the laboratory to select or screen a desired phenotype from mutagenesis. Typically, error-prone polymerase chain reaction (PCR) is used to generate mutant libraries, and DNA shuffling is carried out to recombine existing mutations. It can be performed in a blind manner with limited information on target enzymes, such as structures and reaction mechanisms, but it relies on an effective screening strategy. It is practical for the evolution of carotenogenic enzymes due to the innate traits of carotenoid pigments. Six mutants ((H96L, R203W, A205V, A208V, F213L and A215T) have been isolated to improve the catalytic activity of β-carotene ketolase from Sphingomonas sp. . Three mutations (L175M, M99V, and M99I) of ketolase from Paracoccus sp. result in the improvement if its specificity of to synthesize astaxanthin . Staphylococcus aureus dehydrosqualene (C30) synthase has evolved to synthesize lycopene by mutation F26L or F26S . DNA shuffling of phytoene desaturases from P. agglomerans and P. ananatis results in the isolation of a variant favoring the production of fully conjugated tetradehydrolycopene . Rational design of proteins is based on the in silico simulation and the prediction using a priori enzyme information, which greatly liberates biologists from onerous screening task. This strategy requires adequate information to predict specific targeted amino acid mutations, which can confer desired enzyme traits . Unfortunately, the limited information on carotenogenic enzymes leads to few achievements using such a method.
As aforementioned, carotenoids are derived from the central isoprenoid pathway, which is also employed to synthesize several essential and secondary metabolites in nature. The carotenoid-based colorimetric screening has been developed for evolution of other isoprenoid pathway enzymes. Mutations of GGPP synthase are hypothesized to affect the binding efficiency of the magnesium ions needed for substrate anchoring and improve its catalysis. An error-prone PCR library of Tsuga canadensis GPPS has been screened using the lycopene synthesis pathway as a colorimetric reporter. The GPPS variant (S239C and G295D) is created to increase levopimaradiene production with a 1.7-fold increase over the wild type in E. coli . Augmentation of one pathway can tamper with other pathways, which utilize the same substrate in one organism. Based on this fact, mutagenesis libraries of terpene synthases have been screened by depigmentation of colonies due to the competition between terpene synthases and carotenoid synthases for isoprenyl diphosphates, since the weakened carotenoid color intensity indicates an improvement of terpene synthase activity .
3.5. Development of Microalgae for Carotenoid Production
Algae are a diverse group of aquatic, photosynthetic organisms, generally categorized as macroalgae (i.e., seaweed) and unicellular microalgae. Microalgae have recently garnered interest for production of valuable chemicals including carotenoids [41,131], because they are generally regarded as safe (GRAS) for human consumption and possess the renewable-energy capturing ability of photosynthesis. Moreover, these organisms can be used for genetic manipulation and high-throughput analysis . Some microalgae are also native carotenoid producers (i.e., D. salina for β-carotene and H. pluvialis for astaxanthin). The carotenoid production from microalgae is closely related to culture conditions such as illumination, pH, temperature, nitrogen availability and source, salinity, the oxidant substances, and growth rate [12,133,134]. D. salina is a model species of green microalgae which is widely cultivated outdoors for β-carotene production . A systematic evaluation has been done to decipher the relationship between abiotic stresses (Nitrate concentration, salinity and light quality) and lutein synthesis in D. salina . The abiotic stresses can also be applied to adaptive evolution of microalgae , in a similar manner to strain evolution in yeast for β-carotene production . The freshwater microalga Chlamydomonas reinhardtii is the first and the best studied transformed Chlorophyte, and the nuclear genetic manipulation is easy and well established. It has been engineered with β-carotene ketolase from H. pluvialis to synthesize ketolutein (Hb1H'a1, Figure 3) and adonixanthin (Ha1H'a2, Figure 3) . It is possible to produce diverse valuable carotenoids from marine microalgae with the development of more available genetic tools and technologies.
4. Opportunities and Challenges
The vast and mysterious ocean breeds diverse marine lives and provides unexhausted foodstuffs, nutriment, and drugs for humans. Diverse carotenoids are found from marine species and show broad utilities as colorant fragrance cosmetics and pharmaceuticals. The synthetic pathway of several carotenoids has been illuminated from marine species, which could benefit engineering processes in several host organisms for the production of carotenoids such as β-carotene, astaxanthin, and lutein. On the other hand, carotenoids such as β-carotene often undergo a series of modifications in the miraculous marine ecosphere. And indeed, several novel carotenoids have been isolated during the exploration of the marine ecosphere, while their pharmaceutical potentials remain to be examined due to the limited amount of extracts. Metabolic engineering and synthetic biology allow the assembly of such a chimeric pathway in a tractable organism for the mass production of rare carotenoids and also exhibit the potential to extend the catalogs of carotenoids to non-natural carotenoids, which could accelerate the exploration of novel carotenoids. It is noted that decoded carotenoid pathways and enzymes are still limited to a few marine organisms, although the J. Craig Venter Institute with worldwide collaboration had sequenced and annotated the genomes of 177 marine microbes up until 2010. However, we believe that the developed and developing technologies will allow us to search for novel marine carotenoid pathways in the future.
This work was supported by a grant (NRF-2013R1A1A2008289) from the National Research Foundation, the Intelligent Synthetic Biology Center of Global Frontier Project funded by the MSIP (2011-0031964), and a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ00952003), Rural Development Administration (RDA), Korea. J.K. is supported by scholarships from the BK21 Plus Program, Ministry of Education, Science & Technology (MEST), Korea.
S.K. conceived the idea and held and corrected the manuscript. C.W. and J.K collected the literature, analyzed the data, and wrote the manuscript. C.W. and J.K contributed to this manuscript equally.
Conflicts of Interest
The authors declare no conflict of interest.
- Cazzonelli, C. Carotenoids in nature: Insights from plants and beyond. Funct. Plant Biol. 2011, 38, 833–847. [Google Scholar] [CrossRef]
- Bertrand, M. Carotenoid biosynthesis in diatoms. Photosynth. Res. 2010, 106, 89–102. [Google Scholar] [CrossRef] [PubMed]
- Guedes, A.C.; Amaro, H.M.; Malcata, F.X. Microalgae as sources of carotenoids. Mar. Drugs 2011, 9, 625–644. [Google Scholar] [CrossRef] [PubMed]
- Shindo, K.; Misawa, N. New and rare carotenoids isolated from marine bacteria and their antioxidant activities. Mar. Drugs 2014, 12, 1690–1698. [Google Scholar] [CrossRef] [PubMed]
- Britton, G.; Liaaen-Jensen, S.; Pfander, H. Carotenoids Handbook; Birkhäuser: Basel, Switzerland, 2004. [Google Scholar]
- Armstrong, G.A.; Hearst, J.E. Carotenoids 2: Genetics and molecular biology of carotenoid pigment biosynthesis. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 1996, 10, 228–237. [Google Scholar]
- Mathews, M.M.; Sistrom, W.R. The function of the carotenoid pigments of Sarcina lutea. Archiv. Mikrobiol. 1960, 35, 139–146. [Google Scholar] [CrossRef]
- Liu, J.; Novero, M.; Charnikhova, T.; Ferrandino, A.; Schubert, A.; Ruyter-Spira, C.; Bonfante, P.; Lovisolo, C.; Bouwmeester, H.J.; Cardinale, F. Carotenoid cleavage dioxygenase 7 modulates plant growth, reproduction, senescence, and determinate nodulation in the model legume Lotus japonicus. J. Exp. Bot. 2013, 64, 1967–1981. [Google Scholar] [CrossRef] [PubMed]
- Bolhassani, A.; Khavari, A.; Bathaie, S.Z. Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochim. Biophys. Acta 2014, 1845, 20–30. [Google Scholar]
- Rodrigues, E.; Mariutti, L.R.; Mercadante, A.Z. Scavenging capacity of marine carotenoids against reactive oxygen and nitrogen species in a membrane-mimicking system. Mar. Drugs 2012, 10, 1784–1798. [Google Scholar] [CrossRef] [PubMed]
- Van Den Berg, H.; Faulks, R.; Fernando Granado, H.; Hirschberg, J.; Olmedilla, B.; Sandmann, G.; Southon, S.; Stahl, W. The potential for the improvement of carotenoid levels in foods and the likely systemic effects. J. Sci. Food Agric. 2000, 80, 880–912. [Google Scholar]
- Fernandez-Sevilla, J.M.; Acien Fernandez, F.G.; Molina Grima, E. Biotechnological production of lutein and its applications. Appl. Microbiol. Biotechnol. 2010, 86, 27–40. [Google Scholar]
- Kirsh, V.A.; Mayne, S.T.; Peters, U.; Chatterjee, N.; Leitzmann, M.F.; Dixon, L.B.; Urban, D.A.; Crawford, E.D.; Hayes, R.B. A prospective study of lycopene and tomato product intake and risk of prostate cancer. Cancer Epidemiol. Biomark. Prev. Publ. Am. Assoc. Cancer Res. Cosponsored Am. Soc. Prev. Oncol. 2006, 15, 92–98. [Google Scholar] [CrossRef]
- Wang, W.; Shinto, L.; Connor, W.E.; Quinn, J.F. Nutritional biomarkers in alzheimer’s disease: The association between carotenoids, n-3 fatty acids, and dementia severity. J. Alzheimer’s Dis. JAD 2008, 13, 31–38. [Google Scholar]
- Cosgrove, J. The carotenoid market: Beyond beta-carotene. Nutraceuticals World, 13 December 2010. Available online: http://www.nutraceuticalsworld.com/contents/view_online-exclusives/2010-12-13/the-carotenoid-market-beyond-beta-carotene/ (accessed on 5 September 2014). [Google Scholar]
- Rodriguez-Saiz, M.; de la Fuente, J.L.; Barredo, J.L. Xanthophyllomyces dendrorhous for the industrial production of astaxanthin. Appl. Microbiol. Biotechnol. 2010, 88, 645–658. [Google Scholar]
- Kim, Y.S.; Lee, J.H.; Kim, N.H.; Yeom, S.J.; Kim, S.W.; Oh, D.K. Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered Escherichia coli. Appl. Microbiol. Biotechnol. 2011, 90, 489–497. [Google Scholar] [CrossRef] [PubMed]
- Vilchez, C.; Forjan, E.; Cuaresma, M.; Bedmar, F.; Garbayo, I.; Vega, J.M. Marine carotenoids: Biological functions and commercial applications. Mar. Drugs 2011, 9, 319–333. [Google Scholar] [CrossRef] [PubMed]
- Miziorko, H.M. Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch. Biochem. Biophys. 2011, 505, 131–143. [Google Scholar] [CrossRef] [PubMed]
- Hunter, W.N. The non-mevalonate pathway of isoprenoid precursor biosynthesis. J. Biol. Chem. 2007, 282, 21573–21577. [Google Scholar] [CrossRef] [PubMed]
- Wang, C.; Kim, J.Y.; Choi, E.S.; Kim, S.W. Microbial production of farnesol (FOH): Current states and beyond. Process. Biochem. 2011, 46, 1221–1229. [Google Scholar] [CrossRef]
- Rohdich, F.; Kis, K.; Bacher, A.; Eisenreich, W. The non-mevalonate pathway of isoprenoids: Genes, enzymes and intermediates. Curr. Opin. Chem. Biol. 2001, 5, 535–540. [Google Scholar] [CrossRef] [PubMed]
- Fujisaki, S.; Hara, H.; Nishimura, Y.; Horiuchi, K.; Nishino, T. Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli. J. Biochem. 1990, 108, 995–1000. [Google Scholar] [PubMed]
- Math, S.K.; Hearst, J.E.; Poulter, C.D. The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc. Natl. Acad. Sci. USA 1992, 89, 6761–6764. [Google Scholar] [CrossRef] [PubMed]
- Raisig, A.; Bartley, G.; Scolnik, P.; Sandmann, G. Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli. J. Biochem. 1996, 119, 559–564. [Google Scholar] [CrossRef] [PubMed]
- Garcia-Asuaa, G.; Langa, H.; Cogdellb, R.; Hunter, C.N. Carotenoid diversity: A modular role for the phytoene desaturase step. Trends Plant Sci. 1998, 3, 445–449. [Google Scholar]
- Takaichi, S.; Mochimaru, M. Carotenoids and carotenogenesis in cyanobacteria: Unique ketocarotenoids and carotenoid glycosides. Cell. Mol. Life Sci. CMLS 2007, 64, 2607–2619. [Google Scholar] [CrossRef]
- Takaichi, S. Carotenoids in algae: Distributions, biosyntheses and functions. Mar. Drugs 2011, 9, 1101–1118. [Google Scholar] [CrossRef] [PubMed]
- Umeno, D.; Tobias, A.V.; Arnold, F.H. Diversifying carotenoid biosynthetic pathways by directed evolution. Microbiol. Mol. Biol. Rev. MMBR 2005, 69, 51–78. [Google Scholar] [CrossRef]
- Schaub, P.; Yu, Q.; Gemmecker, S.; Poussin-Courmontagne, P.; Mailliot, J.; McEwen, A.G.; Ghisla, S.; Al-Babili, S.; Cavarelli, J.; Beyer, P. On the structure and function of the phytoene desaturase CrtI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase. PLoS One 2012, 7, e39550. [Google Scholar] [CrossRef] [PubMed]
- Liu, C.I.; Liu, G.Y.; Song, Y.; Yin, F.; Hensler, M.E.; Jeng, W.Y.; Nizet, V.; Wang, A.H.; Oldfield, E. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science 2008, 319, 1391–1394. [Google Scholar] [CrossRef] [PubMed]
- Tobias, A.V.; Arnold, F.H. Biosynthesis of novel carotenoid families based on unnatural carbon backbones: A model for diversification of natural product pathways. Biochim. Biophys. Acta 2006, 1761, 235–246. [Google Scholar] [CrossRef]
- Martin, W.; Rujan, T.; Richly, E.; Hansen, A.; Cornelsen, S.; Lins, T.; Leister, D.; Stoebe, B.; Hasegawa, M.; Penny, D. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. USA 2002, 99, 12246–12251. [Google Scholar] [CrossRef] [PubMed]
- Reyes-Prieto, A.; Hackett, J.D.; Soares, M.B.; Bonaldo, M.F.; Bhattacharya, D. Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions. Curr. Biol. CB 2006, 16, 2320–2325. [Google Scholar]
- Sandmann, G. Evolution of carotene desaturation: The complication of a simple pathway. Arch. Biochem. Biophys. 2009, 483, 169–174. [Google Scholar] [CrossRef] [PubMed]
- Steiger, S.; Jackisch, Y.; Sandmann, G. Carotenoid biosynthesis in Gloeobacter violaceus PCC4721 involves a single CrtI-type phytoene desaturase instead of typical cyanobacterial enzymes. Arch. Microbiol. 2005, 184, 207–214. [Google Scholar] [CrossRef] [PubMed]
- Tsuchiya, T.; Takaichi, S.; Misawa, N.; Maoka, T.; Miyashita, H.; Mimuro, M. The cyanobacterium Gloeobacter violaceus PCC4721 uses bacterial-type phytoene desaturase in carotenoid biosynthesis. FEBS Lett. 2005, 579, 2125–2129. [Google Scholar] [CrossRef] [PubMed]
- Harada, J.; Nagashima, K.V.; Takaichi, S.; Misawa, N.; Matsuura, K.; Shimada, K. Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene. Plant Cell Physiol. 2001, 42, 1112–1118. [Google Scholar] [CrossRef] [PubMed]
- Frigaard, N.U.; Maresca, J.A.; Yunker, C.E.; Jones, A.D.; Bryant, D.A. Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum. J. Bacterial. 2004, 186, 5210–5220. [Google Scholar] [CrossRef]
- Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar]
- Lamers, P.P.; Janssen, M.; de Vos, R.C.; Bino, R.J.; Wijffels, R.H. Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends Biotechnol. 2008, 26, 631–638. [Google Scholar] [CrossRef] [PubMed]
- Rabbani, S.; Beyer, P.; Lintig, J.; Hugueney, P.; Kleinig, H. Induced β-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil. Plant Physiol. 1998, 116, 1239–1248. [Google Scholar] [CrossRef] [PubMed]
- Cui, H.; Yu, X.; Wang, Y.; Cui, Y.; Li, X.; Liu, Z.; Qin, S. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae. BMC Genomics 2013, 14, 457. [Google Scholar] [CrossRef] [PubMed]
- El-Baky, H.A.; BaZ, F.E.; El-Baroty, G. Spirulina species as a source of carotenoids and α-tocopherol and its anticarcinoma factors. Biotechnology 2003, 2, 222–240. [Google Scholar]
- Choubert, G.; Heinrich, O. Carotenoid pigments of the green alga Haematococcus pluvialis: Assay on rainbow trout, Oncorhynchus mykiss, pigmentation in comparison with synthetic astaxanthin and canthaxanthin. Aquaculture 1993, 112, 217–226. [Google Scholar] [CrossRef]
- Abe, K.; Hattori, H.; Hirano, M. Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. Multistriata. Food Chem. 2007, 100, 656–661. [Google Scholar] [CrossRef]
- Sanchez, J.F.; Fernandez-Sevilla, J.M.; Acien, F.G.; Ceron, M.C.; Perez-Parra, J.; Molina-Grima, E. Biomass and lutein productivity of Scenedesmus almeriensis: Influence of irradiance, dilution rate and temperature. Appl. Microbiol. Biotechnol. 2008, 79, 719–729. [Google Scholar] [CrossRef] [PubMed]
- Fietz, S.; Nicklisch, A. Acclimation of the diatom Stephanodiscus neoastraea and the cyanobacterium Planktothrix agardhii to simulated natural light fluctuations. Photosynth. Res. 2002, 72, 95–106. [Google Scholar] [CrossRef] [PubMed]
- Takaichi, S. Distributions, biosyntheses and functions of carotenoids in algae. Agro FOOD Industry Hi Tech. 2013, 24, 55–58. [Google Scholar]
- Kupper, H.; Seibert, S.; Parameswaran, A. Fast, sensitive, and inexpensive alternative to analytical pigment HPLC: Quantification of chlorophylls and carotenoids in crude extracts by fitting with Gauss peak spectra. Anal. Chem. 2007, 79, 7611–7627. [Google Scholar] [CrossRef] [PubMed]
- Miyashita, K.; Nishikawa, S.; Beppu, F.; Tsukui, T.; Abe, M.; Hosokawa, M. The allenic carotenoid fucoxanthin, a novel marine nutraceutical from brown seaweeds. J. Sci. Food Agric. 2011, 91, 1166–1174. [Google Scholar] [CrossRef] [PubMed]
- Aitzetmüller, K.; Strain, H.H.; Svec, W.A.; Grandolfo, M.; Katz, J.J. Loroxanthin, a unique xanthophyll from Scenedesmus obliquus and Chlorella vulgaris. Phytochemistry 1969, 1761–1770. [Google Scholar]
- Ganesan, P.; Matsubara, K.; Ohkubo, T.; Tanaka, Y.; Noda, K.; Sugawara, T.; Hirata, T. Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile. Phytomed. Int. J. Phytother. Phytopharmacol. 2010, 17, 1140–1144. [Google Scholar] [CrossRef]
- Maresca, J.A.; Romberger, S.P.; Bryant, D.A. Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases. J. Bacterial. 2008, 190, 6384–6391. [Google Scholar] [CrossRef]
- Brocks, J.J.; Love, G.D.; Summons, R.E.; Knoll, A.H.; Logan, G.A.; Bowden, S.A. Biomarker evidence for green and purple sulphur bacteria in a stratified Palaeoproterozoic sea. Nature 2005, 437, 866–870. [Google Scholar] [CrossRef] [PubMed]
- Graham, J.E.; Bryant, D.A. The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002. J. Bacterial. 2009, 191, 3292–3300. [Google Scholar]
- Song, P.S.; Koka, P.; Prezelin, B.B.; Haxo, F.T. Molecular topology of the photosynthetic light-harvesting pigment complex, peridinin-chlorophyll A-protein, from marine dinoflagellates. Biochemistry 1976, 15, 4422–4427. [Google Scholar] [CrossRef] [PubMed]
- Maoka, T.; Hashimoto, K.; Akimoto, N.; Fujiwara, Y. Structures of five new carotenoids from the oyster Crassostrea gigas. J. Nat. Prod. 2001, 64, 578–581. [Google Scholar] [CrossRef] [PubMed]
- Maoka, T.; Akimoto, N.; Yim, M.J.; Hosokawa, M.; Miyashita, K. New C37 skeletal carotenoid from the clam, Paphia amabillis. J. Agric. Food Chem. 2008, 56, 12069–12072. [Google Scholar] [CrossRef] [PubMed]
- Maoka, T.; Fujiwara, Y.; Hashimoto, K.; Akimoto, N. Structure of new carotenoids from corbicula clam Corbicula japonica. J. Nat. Prod. 2005, 68, 1341–1344. [Google Scholar] [CrossRef] [PubMed]
- Maoka, T. Carotenoids in marine animals. Mar. Drugs 2011, 9, 278–293. [Google Scholar] [CrossRef] [PubMed]
- Maresca, J.A.; Graham, J.E.; Wu, M.; Eisen, J.A.; Bryant, D.A. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria. Proc. Natl. Acad. Sci. USA 2007, 104, 11784–11789. [Google Scholar] [CrossRef] [PubMed]
- Misawa, N. Carotenoid beta-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls. Mar. Drugs 2011, 9, 757–771. [Google Scholar] [CrossRef] [PubMed]
- Kim, J.; DellaPenna, D. Defining the primary route for lutein synthesis in plants: The role of Arabidopsis carotenoid β-ring hydroxylase CYP97A3. Proc. Natl. Acad. Sci. USA 2006, 103, 3474–3479. [Google Scholar] [CrossRef] [PubMed]
- Coesel, S.; Obornik, M.; Varela, J.; Falciatore, A.; Bowler, C. Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms. PLoS One 2008, 3, e2896. [Google Scholar] [CrossRef] [PubMed]
- Mikami, K.; Hosokawa, M. Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds. Int. J. Mol. Sci. 2013, 14, 13763–13781. [Google Scholar] [CrossRef] [PubMed]
- Yang, L.E.; Huang, X.Q.; Hang, Y.; Deng, Y.Y.; Lu, Q.Q.; Lu, S. The P450-type carotene hydroxylase PuCHY1 from Porphyra suggested the evolution of carotenoid metabolism in red algae. J. Integr. Plant Boil. 2014. in press. [Google Scholar]
- Misawa, N.; Satomi, Y.; Kondo, K.; Yokoyama, A.; Kajiwara, S.; Saito, T.; Ohtani, T.; Miki, W. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 1995, 177, 6575–6584. [Google Scholar] [PubMed]
- Netzer, R.; Stafsnes, M.H.; Andreassen, T.; Goksoyr, A.; Bruheim, P.; Brautaset, T. Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: Heterologous expression and evidence for diverse and multiple catalytic functions of C50 carotenoid cyclases. J. Bacteriol. 2010, 192, 5688–5699. [Google Scholar] [CrossRef] [PubMed]
- Zhang, W.; Hu, X.; Wang, L.; Wang, X. Reconstruction of the carotenoid biosynthetic pathway of Cronobacter sakazakii BAA894 in Escherichia coli. PLoS One 2014, 9, e86739. [Google Scholar] [CrossRef] [PubMed]
- Kim, S.W.; Keasling, J.D. Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol. Bioeng. 2001, 72, 408–415. [Google Scholar] [CrossRef] [PubMed]
- Harker, M.; Bramley, P.M. Expression of prokaryotic 1-deoxy-d-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis. FEBS Lett. 1999, 448, 115–119. [Google Scholar] [CrossRef] [PubMed]
- Wang, G.S.; Grammel, H.; Abou-Aisha, K.; Sagesser, R.; Ghosh, R. High-Level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum. Appl. Environ. Microbiol. 2012, 78, 7205–7215. [Google Scholar] [CrossRef] [PubMed]
- Miura, Y.; Kondo, K.; Saito, T.; Shimada, H.; Fraser, P.D.; Misawa, N. Production of the carotenoids lycopene, β-carotene, and astaxanthin in the food yeast Candida utilis. Appl. Environ. Microbiol. 1998, 64, 1226–1229. [Google Scholar] [PubMed]
- Harada, H.; Misawa, N. Novel approaches and achievements in biosynthesis of functional isoprenoids in Escherichia coli. Appl. Microbiol. Biotechnol. 2009, 84, 1021–1031. [Google Scholar] [CrossRef] [PubMed]
- Yamano, S.; Ishii, T.; Nakagawa, M.; Ikenaga, H.; Misawa, N. Metabolic engineering for production of β-carotene and lycopene in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 1994, 58, 1112–1114. [Google Scholar] [CrossRef] [PubMed]
- Heider, S.A.; Peters-Wendisch, P.; Netzer, R.; Stafnes, M.; Brautaset, T.; Wendisch, V.F. Production and glucosylation of C50 and C40 carotenoids by metabolically engineered Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 2014, 98, 1223–1235. [Google Scholar] [CrossRef] [PubMed]
- Yoon, S.H.; Kim, J.E.; Lee, S.H.; Park, H.M.; Choi, M.S.; Kim, J.Y.; Shin, Y.C.; Keasling, J.D.; Kim, S.W. Engineering the lycopene synthetic pathway in E. coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis. Appl. Microbiol. Biotechnol. 2007, 74, 131–139. [Google Scholar]
- Sandmann, G.; Albrecht, M.; Schnurr, G.; Knorzer, O.; Boger, P. The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli. Trends Biotechnol. 1999, 17, 233–237. [Google Scholar] [CrossRef] [PubMed]
- Misawa, N. Pathway engineering for functional isoprenoids. Curr. Opin. Biotechnol. 2011, 22, 627–633. [Google Scholar] [CrossRef] [PubMed]
- Yoon, S.H.; Park, H.M.; Kim, J.E.; Lee, S.H.; Choi, M.S.; Kim, J.Y.; Oh, D.K.; Keasling, J.D.; Kim, S.W. Increased β-carotene production in recombinant Escherichia coli harboring an engineered isoprenoid precursor pathway with mevalonate addition. Biotechnol. Prog. 2007, 23, 599–605. [Google Scholar] [CrossRef] [PubMed]
- Verwaal, R.; Wang, J.; Meijnen, J.P.; Visser, H.; Sandmann, G.; van den Berg, J.A.; van Ooyen, A.J. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 2007, 73, 4342–4350. [Google Scholar] [CrossRef] [PubMed]
- Yoon, S.H.; Lee, S.H.; Das, A.; Ryu, H.K.; Jang, H.J.; Kim, J.Y.; Oh, D.K.; Keasling, J.D.; Kim, S.W. Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. J. Biotechnol. 2009, 140, 218–226. [Google Scholar] [CrossRef]
- Yoshida, K.; Ueda, S.; Maeda, I. Carotenoid production in Bacillus subtilis achieved by metabolic engineering. Biotechnol. Lett. 2009, 31, 1789–1793. [Google Scholar] [CrossRef] [PubMed]
- Beuttler, H.; Hoffmann, J.; Jeske, M.; Hauer, B.; Schmid, R.D.; Altenbuchner, J.; Urlacher, V.B. Biosynthesis of zeaxanthin in recombinant Pseudomonas putida. Appl. Microbiol. Biotechnol. 2011, 89, 1137–1147. [Google Scholar] [CrossRef] [PubMed]
- Ye, V.M.; Bhatia, S.K. Pathway engineering strategies for production of beneficial carotenoids in microbial hosts. Biotechnol. Lett. 2012, 34, 1405–1414. [Google Scholar] [CrossRef] [PubMed]
- Vick, J.E.; Johnson, E.T.; Choudhary, S.; Bloch, S.E.; Lopez-Gallego, F.; Srivastava, P.; Tikh, I.B.; Wawrzyn, G.T.; Schmidt-Dannert, C. Optimized compatible set of Biobrick vectors for metabolic pathway engineering. Appl. Microbiol. Biotechnol. 2011, 92, 1275–1286. [Google Scholar] [CrossRef] [PubMed]
- Li, M.Z.; Elledge, S.J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 2007, 4, 251–256. [Google Scholar] [CrossRef] [PubMed]
- Gibson, D.G.; Young, L.; Chuang, R.Y.; Venter, J.C.; Hutchison, C.A., 3rd; Smith, H.O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, 343–345. [Google Scholar]
- Wingler, L.M.; Cornish, V.W. Reiterative recombination for the in vivo assembly of libraries of multigene pathways. Proc. Natl. Acad. Sci. USA 2011, 108, 15135–15140. [Google Scholar] [CrossRef] [PubMed]
- Sleight, S.C.; Sauro, H.M. Randomized Biobrick assembly: A novel DNA assembly method for randomizing and optimizing genetic circuits and metabolic pathways. ACS Synth. Boil. 2013, 2, 506–518. [Google Scholar] [CrossRef]
- Yadav, V.G.; de Mey, M.; Lim, C.G.; Ajikumar, P.K.; Stephanopoulos, G. The future of metabolic engineering and synthetic biology: Towards a systematic practice. Metab. Eng. 2012, 14, 233–241. [Google Scholar] [CrossRef] [PubMed]
- Ajikumar, P.K.; Xiao, W.H.; Tyo, K.E.; Wang, Y.; Simeon, F.; Leonard, E.; Mucha, O.; Phon, T.H.; Pfeifer, B.; Stephanopoulos, G. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science 2010, 330, 70–74. [Google Scholar] [CrossRef] [PubMed]
- Temme, K.; Hill, R.; Segall-Shapiro, T.H.; Moser, F.; Voigt, C.A. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res. 2012, 40, 8773–8781. [Google Scholar] [CrossRef] [PubMed]
- Salis, H.M.; Mirsky, E.A.; Voigt, C.A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 2009, 27, 946–950. [Google Scholar] [CrossRef] [PubMed]
- Pfleger, B.F.; Pitera, D.J.; Smolke, C.D.; Keasling, J.D. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 2006, 24, 1027–1032. [Google Scholar] [CrossRef] [PubMed]
- Qi, L.; Haurwitz, R.E.; Shao, W.; Doudna, J.A.; Arkin, A.P. RNA processing enables predictable programming of gene expression. Nat. Biotechnol. 2012, 30, 1002–1006. [Google Scholar] [CrossRef] [PubMed]
- Dueber, J.E.; Wu, G.C.; Malmirchegini, G.R.; Moon, T.S.; Petzold, C.J.; Ullal, A.V.; Prather, K.L.; Keasling, J.D. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 2009, 27, 753–759. [Google Scholar] [CrossRef] [PubMed]
- Parsons, J.B.; Dinesh, S.D.; Deery, E.; Leech, H.K.; Brindley, A.A.; Heldt, D.; Frank, S.; Smales, C.M.; Lunsdorf, H.; Rambach, A.; et al. Biochemical and structural insights into bacterial organelle form and biogenesis. J. Biol. Chem. 2008, 283, 14366–14375. [Google Scholar] [CrossRef] [PubMed]
- Martin, V.J.; Pitera, D.J.; Withers, S.T.; Newman, J.D.; Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 2003, 21, 796–802. [Google Scholar] [CrossRef] [PubMed]
- Zhang, F.; Carothers, J.M.; Keasling, J.D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat. Biotechnol. 2012, 30, 354–359. [Google Scholar] [CrossRef] [PubMed]
- Farmer, W.R.; Liao, J.C. Improving lycopene production in Escherichia coli by engineering metabolic control. Nat. Biotechnol. 2000, 18, 533–537. [Google Scholar] [CrossRef] [PubMed]
- Farmer, W.R.; Liao, J.C. Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol. Prog. 2001, 17, 57–61. [Google Scholar] [CrossRef] [PubMed]
- Zhou, Y.; Nambou, K.; Wei, L.; Cao, J.; Imanaka, T.; Hua, Q. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase. Biotechnol. Lett. 2013, 35, 2137–2145. [Google Scholar] [CrossRef] [PubMed]
- Zhang, C.; Chen, X.; Zou, R.; Zhou, K.; Stephanopoulos, G.; Too, H.P. Combining genotype improvement and statistical media optimization for isoprenoid production in E. coli. PLoS One 2013, 8, e75164. [Google Scholar] [CrossRef]
- Suh, W. High isoprenoid flux Escherichia coli as a host for carotenoids production. Methods Mol. Boil. 2012, 834, 49–62. [Google Scholar]
- Reyes, L.H.; Gomez, J.M.; Kao, K.C. Improving carotenoids production in yeast via adaptive laboratory evolution. Metab. Eng. 2014, 21, 26–33. [Google Scholar] [CrossRef] [PubMed]
- Ozaydin, B.; Burd, H.; Lee, T.S.; Keasling, J.D. Carotenoid-Based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production. Metab. Eng. 2013, 15, 174–183. [Google Scholar] [CrossRef] [PubMed]
- Alper, H.; Miyaoku, K.; Stephanopoulos, G. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat. Biotechnol. 2005, 23, 612–616. [Google Scholar]
- Alper, H.; Jin, Y.S.; Moxley, J.F.; Stephanopoulos, G. Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab. Eng. 2005, 7, 155–164. [Google Scholar] [CrossRef] [PubMed]
- Choi, H.S.; Lee, S.Y.; Kim, T.Y.; Woo, H.M. In silico identification of gene amplification targets for improvement of lycopene production. Appl. Environ. Microbiol. 2010, 76, 3097–3105. [Google Scholar] [CrossRef] [PubMed]
- Kang, M.J.; Lee, Y.M.; Yoon, S.H.; Kim, J.H.; Ock, S.W.; Jung, K.H.; Shin, Y.C.; Keasling, J.D.; Kim, S.W. Identification of genes affecting lycopene accumulation in Escherichia coli using a shot-gun method. Biotechnol. Bioeng. 2005, 91, 636–642. [Google Scholar] [CrossRef] [PubMed]
- Alper, H.; Stephanopoulos, G. Uncovering the gene knockout landscape for improved lycopene production in E. coli. Appl. Microbiol. Biotechnol. 2008, 78, 801–810. [Google Scholar] [CrossRef]
- Jin, Y.S.; Stephanopoulos, G. Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli. Metab. Eng. 2007, 9, 337–347. [Google Scholar] [CrossRef] [PubMed]
- Zhao, J.; Li, Q.; Sun, T.; Zhu, X.; Xu, H.; Tang, J.; Zhang, X.; Ma, Y. Engineering central metabolic modules of Escherichia coli for improving β-carotene production. Metab. Eng. 2013, 17, 42–50. [Google Scholar] [CrossRef] [PubMed]
- Wang, H.H.; Isaacs, F.J.; Carr, P.A.; Sun, Z.Z.; Xu, G.; Forest, C.R.; Church, G.M. Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460, 894–898. [Google Scholar] [CrossRef] [PubMed]
- Warner, J.R.; Reeder, P.J.; Karimpour-Fard, A.; Woodruff, L.B.; Gill, R.T. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat. Biotechnol. 2010, 28, 856–862. [Google Scholar] [CrossRef] [PubMed]
- Kachroo, A.H.; Jayaram, M.; Rowley, P.A. Metabolic engineering without plasmids. Nat. Biotechnol. 2009, 27, 729–731. [Google Scholar] [CrossRef] [PubMed]
- Chiang, C.J.; Chen, P.T.; Chao, Y.P. Replicon-free and markerless methods for genomic insertion of dnas in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli. Biotechnol. Bioeng. 2008, 101, 985–995. [Google Scholar] [CrossRef] [PubMed]
- Chen, Y.Y.; Shen, H.J.; Cui, Y.Y.; Chen, S.G.; Weng, Z.M.; Zhao, M.; Liu, J.Z. Chromosomal evolution of Escherichia coli for the efficient production of lycopene. BMC Biotechnol. 2013, 13, 6. [Google Scholar] [CrossRef] [PubMed]
- Shin, J.H.; Kim, H.U.; Kim, D.I.; Lee, S.Y. Production of bulk chemicals via novel metabolic pathways in microorganisms. Biotechnol. Adv. 2013, 31, 925–935. [Google Scholar] [CrossRef] [PubMed]
- Zanghellini, A. De novo computational enzyme design. Curr. Opin. Biotechnol. 2014, 29C, 132–138. [Google Scholar] [CrossRef] [PubMed]
- Damborsky, J.; Brezovsky, J. Computational tools for designing and engineering enzymes. Curr. Opin. Chem. Biol. 2014, 19C, 8–16. [Google Scholar] [CrossRef]
- Tao, L.; Wilczek, J.; Odom, J.M.; Cheng, Q. Engineering a β-carotene ketolase for astaxanthin production. Metab. Eng. 2006, 8, 523–531. [Google Scholar] [CrossRef] [PubMed]
- Ye, R.W.; Stead, K.J.; Yao, H.; He, H. Mutational and functional analysis of the β-carotene ketolase involved in the production of canthaxanthin and astaxanthin. Appl. Environ. Microbiol. 2006, 72, 5829–5837. [Google Scholar] [CrossRef] [PubMed]
- Umeno, D.; Tobias, A.V.; Arnold, F.H. Evolution of the C30 carotenoid synthase CrtM for function in a C40 pathway. J. Bacteriol. 2002, 184, 6690–6699. [Google Scholar] [CrossRef] [PubMed]
- Schmidt-Dannert, C.; Umeno, D.; Arnold, F.H. Molecular breeding of carotenoid biosynthetic pathways. Nat. Biotechnol. 2000, 18, 750–753. [Google Scholar]
- Fuxreiter, M.; Mones, L. The role of reorganization energy in rational enzyme design. Curr. Opin. Chem. Biol. 2014, 21C, 34–41. [Google Scholar] [CrossRef] [PubMed]
- Leonard, E.; Ajikumar, P.K.; Thayer, K.; Xiao, W.H.; Mo, J.D.; Tidor, B.; Stephanopoulos, G.; Prather, K.L. Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc. Natl. Acad. Sci. USA 2010, 107, 13654–13659. [Google Scholar] [CrossRef] [PubMed]
- Furubayashi, M.; Ikezumi, M.; Kajiwara, J.; Iwasaki, M.; Fujii, A.; Li, L.; Saito, K.; Umeno, D. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption. PLoS One 2014, 9, e93317. [Google Scholar] [CrossRef] [PubMed]
- Del Campo, J.A.; Garcia-Gonzalez, M.; Guerrero, M.G. Outdoor cultivation of microalgae for carotenoid production: Current state and perspectives. Appl. Microbiol. Biotechnol. 2007, 74, 1163–1174. [Google Scholar]
- Rosenberg, J.N.; Oyler, G.A.; Wilkinson, L.; Betenbaugh, M.J. A green light for engineered algae: Redirecting metabolism to fuel a biotechnology revolution. Curr. Opin. Biotechnol. 2008, 19, 430–436. [Google Scholar] [CrossRef] [PubMed]
- Binti Ibnu Rasid, E.N.; Mohamad, S.E.; Jamaluddin, H.; Salleh, M.M. Screening factors influencing the production of astaxanthin from freshwater and marine microalgae. Appl. Biochem. Biotechnol. 2014, 172, 2160–2174. [Google Scholar]
- Casal, C.; Cuaresma, M.; Vega, J.M.; Vilchez, C. Enhanced productivity of a lutein-enriched novel acidophile microalga grown on urea. Mar. Drugs 2011, 9, 29–42. [Google Scholar] [CrossRef]
- Fu, W.; Paglia, G.; Magnusdottir, M.; Steinarsdottir, E.A.; Gudmundsson, S.; Palsson, B.O.; Andresson, O.S.; Brynjolfsson, S. Effects of abiotic stressors on lutein production in the green microalga Dunaliella salina. Microb. Cell Factories 2014, 13, 3. [Google Scholar] [CrossRef]
- Fu, W.; Guethmundsson, O.; Paglia, G.; Herjolfsson, G.; Andresson, O.S.; Palsson, B.O.; Brynjolfsson, S. Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution. Appl. Microbiol. Biotechnol. 2013, 97, 2395–2403. [Google Scholar] [CrossRef] [PubMed]
- Leon, R.; Couso, I.; Fernandez, E. Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. J. Biotechnol. 2007, 130, 143–152. [Google Scholar] [CrossRef] [PubMed]
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).