Genes and Pathway Reactions Related to Carotenoid Biosynthesis in Purple Bacteria.

In purple bacteria, the genes of the carotenoid pathways are part of photosynthesis gene clusters which were distributed among different species by horizontal gene transfer. Their close organisation facilitated the first-time cloning of carotenogenic genes and promoted the molecular investigation of spheroidene and spirilloxanthin biosynthesis. This review highlights the cloning of the spheroidene and spirilloxanthin pathway genes and presents the current knowledge on the enzymes involved in the carotenoid biosynthesis of purple sulphur and non-sulphur bacteria. Mostly, spheroidene or spirilloxanthin biosynthesis exists in purple non-sulphur bacteria but both pathways operate simultaneously in Rubrivivax gelatinosus. In the following years, genes from other bacteria including purple sulphur bacteria with an okenone pathway were cloned. The individual steps were investigated by kinetic studies with heterologously expressed pathway genes which supported the establishment of the reaction mechanisms. In particular, the substrate and product specificities revealed the sequential order of the speroidene and spiriloxanthin pathways as well as their interactions. Information on the enzymes involved revealed that the phytoene desaturase determines the type of pathway by the formation of different products. By selection of mutants with amino acid exchanges in the putative substrate-binding site, the neurosporene-forming phytoene desaturase could be changed into a lycopene-producing enzyme and vice versa. Concerning the oxygen groups in neurosporene and lycopene, the tertiary alcohol group at C1 is formed from water and not by oxygenation, and the C2 or C4 keto groups are inserted differently by an oxygen-dependent or oxygen-independent ketolation reaction, respectively.

Generation of stable homozygous transformants of diploid yeasts such as Xanthophyllomyces dendrorhous.

The nonconventional yeast Xanthophyllomyces dendrorhous is an established platform for genetic pathway modification. A genetic tool box is available and can be used extensively to select from for different engineering strategies. Due to the diploid nature of X. dendrorhous, genetic transformation typically results in heterozygous lines. They are genetically unstable and lose their phenotypes caused by mitotic recombination. In addition, targeted integration for inactivation of genes of the carotenoid pathway resulted in an intermediary phenotype of incomplete pathway disruption. This issue is the main scope of this review. It is illustrated by using genetic modification of the carotenoid pathway of X. dendrorhous as a model system with a focus on the demonstration of how to solve these problems by generation of homozygous lines. They can be selected from heterozygous transformants after spontaneous mitotic recombination and selection or after induced meiotic recombination. Corresponding methods of how to proceed including the initiation of a sexual cycle are described. The selected segregated lines are stable in fermenter cultures without the need of selection pressure. This is an essential requirement for any industrial application. KEY POINTS: • Genetic interventions of diploid yeasts result in heterozygous transformants that are unstable without selection pressure. • This is due to mitotic recombination leading to the elimination of inserted DNA. • Stable homozygous lines can be obtained and selected after either meiotic or mitotic recombination.

Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids.

Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, ß-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of ß-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.

HPLC analysis of carotenoids from bacteria.

Bacteria synthesize different carotenoid structures. A powerful method for the determination of carotenoid profiles of bacteria, including quantification, is high performance liquid chromatography (HPLC). This chapter explains the basics of this method and its application, and provides instruction on how to proceed with HPLC. A basic HPLC system for bacterial carotenoids is presented which allows tentative identification and quantification. The individual analysis steps including extraction, sample preparation and HPLC separation are described in detail. HPLC examples of structurally diverse carotenoids from different bacterial phyla are displayed demonstrating the applicability of this method. In addition, information on spectroscopic properties useful for identification is included. Finally, a procedure for carotenoid quantification from the HPLC chromatogram is described.

Carotenoids and Their Biosynthesis in Fungi.

Carotenoids represent a class of pigmented terpenoids. They are distributed in all taxonomic groups of fungi. Most of the fungal carotenoids differ in their chemical structures to those from other organisms. The general function of carotenoids in heterotrophic organisms is protection as antioxidants against reactive oxygen species generated by photosensitized reactions. Furthermore, carotenoids are metabolized to apocarotenoids by oxidative cleavage. This review presents the current knowledge on fungal-specific carotenoids, their occurrence in different taxonomic groups, and their biosynthesis and conversion into trisporic acids. The outline of the different pathways was focused on the reactions and genes involved in not only the known pathways, but also suggested the possible mechanisms of reactions, which may occur in several non-characterized pathways in different fungi. Finally, efforts and strategies for genetic engineering to enhance or establish pathways for the production of various carotenoids in carotenogenic or non-carotenogenic yeasts were highlighted, addressing the most-advanced producers of each engineered yeast, which offered the highest biotechnological potentials as production systems.

Diversity and origin of carotenoid biosynthesis: its history of coevolution towards plant photosynthesis.

The development of photosynthesis was a highlight in the progression of bacteria. In addition to the photosystems with their structural proteins, the photosynthesis apparatus consists of different cofactors including essential carotenoids. Thus, the evolution of the carotenoid pathways in relation to the functionality of the resulting structures in photosynthesis is the focus of this review. Analysis of carotenoid pathway genes indicates early evolutionary roots in prokaryotes. The pathway complexity leading to a multitude of structures is a result of gene acquisition, including their functional modifications, emergence of novel genes and gene exchange between species. Along with the progression of photosynthesis, carotenoid pathways coevolved with photosynthesis according to their advancing functionality. Cyanobacteria, with their oxygenic photosynthesis, became a landmark for evolutionary events including carotenogenesis. Concurrent with endosymbiosis, the cyanobacterial carotenoid pathways were inherited into algal plastids. In the lineage leading to Chlorophyta and plants, carotenoids evolved to their prominent role in protection and regulation of light energy input as constituents of a highly efficient light-harvesting complex.

Diversity and Evolution of Carotenoid Biosynthesis from Prokaryotes to Plants.

Carotenoids exist in pro- and eukaryotic organisms, but not in animals (with one exception). Their biosynthesis evolved from a common ancestor of Archaea and Bacteria and via the latter by endosymbiosis to algae and plants. The formation of carotenoids in fungi can be regarded as a lineage from the archaea. This review highlights the distribution and evolution of carotenogenic pathways in taxonomic groups of prokaryotes and eukaryotes with a special emphasis on the evolutionary aspects of prominent carotenogenic genes in relation to the assigned function of their corresponding enzymes. The latter aspect includes a focus on paralogs of gene families evolving novel functions and unrelated genes encoding enzymes with the same function.

Engineered Maize Hybrids with Diverse Carotenoid Profiles and Potential Applications in Animal Feeding.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of ß-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high ß:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low ß:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a ß-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic ß-carotene ketolase with a superior ß-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Xanthophyllomyces dendrorhous, a Versatile Platform for the Production of Carotenoids and Other Acetyl-CoA-Derived Compounds.

Xanthophyllomyces dendrorhous (with Phaffia rhodozyma as its anamorphic state) is a basidiomycetous, moderately psychrophilic, red yeast belonging to the Cystofilobasidiales. Its red pigmentation is caused by the accumulation of astaxanthin, which is a unique feature among fungi. The present chapter reviews astaxanthin biosynthesis and acetyl-CoA metabolism in X. dendrorhous and describes the construction of a versatile platform for the production of carotenoids, such as astaxanthin, and other acetyl-CoA-derived compounds including fatty acids by using this fungus.

Carotenoid Biosynthesis in the Phylum Actinobacteria.

Actinobacteria is the phylum that has the biggest genome in the Bacteria domain and includes many colored species. Their pigment analysis revealed that structurally diverse carotenoids are responsible for their pigmentation. This chapter reviews the biosynthesis of the diverse carotenoids of Actinobacteria. Its carotenoids belong to three different types: 1) carotenoid of C50 chain length, 2) carotenoids with aromatic end groups, and 3) keto carotenoid like canthaxanthin (ß,ß-carotene-4,4'-dione) or monocyclic keto-γ-carotene derivatives. Species from the genus Rhodococcus are the only known Actinobacteria with a simultaneous pathway to aromatic and to keto carotenoids.

Carotenoid Production in Escherichia coli: Case of Acyclic Carotenoids.

Among isoprenoids, carotenoids were the first group of compounds which were synthesized from foreign genes in non-carotenogenic Escherichia coli as a heterologous host. A great variety of carotenoids have been shown to be produced in E. coli due to the introduction of combinations of carotenoid biosynthesis genes, which were isolated from carotenogenic organisms. Carotenoids that have been produced in E. coli are mostly cyclic carotenoids that retain carbon 40 (C40) basic structure, except for acyclic carotene lycopene. On the other hand, acyclic carotenoids, which can also be produced in E. coli, comprise a group of carotenoids with diverse chain lengths, i.e., with C20, C30, C40, or C50 basic skeleton. As for acyclic C30, C40, and C50 carotenoids, carotenogenic genes of bacterial origin were needed, while a cleavage dioxygenase gene of higher-plant origin was utilized for the synthesis of acyclic C20 carotenoids. The present chapter is a review on the biosynthesis of such diverse acyclic carotenoids at the gene level.

Nitrogen inputs influence vegetative metabolism in maize engineered with a seed-specific carotenoid pathway.

KEY MESSAGE: Metabolomic profiling of a maize line engineered with an endosperm-specific carotenogenic pathway revealed unexpected metabolic readjustments of primary metabolism in leaves and roots. High-carotenoid (HC) maize was engineered to accumulate high levels of carotenoids in the endosperm. The metabolic interventions influenced the flux through non-target pathways in tissues that were not affected by the targeted intervention. HC maize at the vegetative stage also showed a reduced susceptibility to insect feeding. It is unknown, however, whether the metabolic history of the embryo has any impact on the metabolite composition in vegetative tissues. We, therefore, compared HC maize and its isogenic counterpart (M37W) to test the hypothesis that boosting the carotenoid content in the endosperm triggers compensatory effects in core metabolism in vegetative tissues. Specifically, we investigated whether the metabolite composition of leaves and roots at the V6 stage differs between HC and M37W, and whether N inputs further alter the core metabolism of HC compared to M37W. We found an increase in the abundance of organic acids from the tricarboxylic acid (TCA) cycle in HC even under restricted N conditions. In contrast, low levels of carotenoids and chlorophyll were measured regardless of N levels. Sugars were also significantly depleted in HC under low N. We propose a model explaining the observed genotype-dependent and input-dependent effects, in which organic acids derived from the TCA cycle accumulate during vegetative growth and contribute to the increased demand for pyruvate and/or acetyl-CoA in the endosperm and embryo. This response may in part reflect the transgenerational priming of vegetative tissues in the embryo induced by the increased demand for metabolic precursors during seed development in the previous generation.

Enhanced Coproduction of Cell-Bound Zeaxanthin and Secreted Exopolysaccharides by Sphingobium sp. via Metabolic Engineering and Optimized Fermentation.

Zeaxanthin is a value-added carotenoid with wide applications. This study aims to manipulate a generally recognized as safe and carotenoid-producing bacterium, Sphingobium sp., for enhanced production of zeaxanthin and exopolysaccharides. First, whole-genome sequencing and analysis of pathway genes were applied to define the carotenoid pathway in Sphingobium sp. Second, a Sphingobium transformation system was established to engineer metabolite flux into zeaxanthin. By a combination of chemical mutagenesis and removal of bottlenecks of carotenoid biosynthesis via overexpression of three rate-limiting enzymes, the genetically modified Sphingobium DIZ strain produced 21.26 mg/g dry cell weight of zeaxanthin, which was about 4-fold higher than the wild type. Upon optimization of culture conditions, the DIZ strain produced 479.5 mg/L of zeaxanthin with the productivity of 4.99 mg/L/h and 21.9 g/L of exopolysaccharides using a fed-batch fermentation strategy. This study represents the first genetic manipulation of Sphingobium sp., a biotechnologically important bacterium, for high-yield production of value-added metabolites.

Antioxidant Protection from UV- and Light-Stress Related to Carotenoid Structures.

This review summarizes studies of protection against singlet oxygen and radical damage by carotenoids. The main focus is on how substitutions of the carotenoid molecules determine high antioxidant activities such as singlet oxygen quenching and radical scavenging. Applied assays were carried out either in vitro in solvents or with liposomes, and in a few cases with living organisms. In the latter, protection by carotenoids especially of photosynthesis against light- and UV-stress is of major importance, but also heterotrophic organisms suffer from high light and UV exposure which can be alleviated by carotenoids. Carotenoids to be compared include C30, C40 and C50 molecules either acyclic, monocyclic or bicyclic with different substitutions including sugar and fatty acid moieties. Although some studies are difficult to compare, there is a tendency towards mono and bicyclic carotenoids with keto groups at C-4/C-4' and the longest possible polyene structure functions to act best in singlet oxygen quenching and radical scavenging. Size of the carotenoid and lipophilic substituents such as fatty acids seem to be of minor importance for their activity but hydroxyl groups at an acyclic end and especially glycosylation of these hydroxyl groups enhance carotenoid activity.

Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini.

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.

Genetic modification of the carotenoid pathway in the red yeast Xanthophyllomyces dendrorhous: Engineering of a high-yield zeaxanthin strain.

The red yeast Xanthophyllomyces dendrorhous was genetically engineered for high-yield accumulation of the carotenoid zeaxanthin. Initially, an astaxanthin hyper-producing mutant was used to generate a ß-carotene synthesizing transformant by inactivation of the astaxanthin synthase gene. Subsequently, a bacterial ß-carotene hydroxylase gene was genome integrated to establish ß-carotene to zeaxanthin conversion. Crucial for efficient zeaxanthin formation was the rate of this hydroxylation which was related to the number of integrated gene copies. Two strategies were followed to get multiple integrations, either random integration into the ribosomal DNA which resulted in a maximum copy number of 10, or directly integration of a total of 8 copies into both alleles of the astaxanthin synthase gene. Combining both procedures with additional insertion of the gene to enhance expression of the carotenogenesis limiting phytoene synthase, a transformant reaching a high level of zeaxanthin of 5.2 mg/g dw was finally generated. The application of pentose sugars including xylose as substrates for X. dendrorhous which avoids the inhibitory Crab-tree effect of glucose is favorable for carotenogenesis allowing the replacement of glucose by a hydrolysate of the waste product hemicellulose which is rich in xylose demonstrating ithe effectiveness as a sustainable and cost-efficient alternative for high-yield zeaxanthin formation.

The Silencing of Carotenoid ß-Hydroxylases by RNA Interference in Different Maize Genetic Backgrounds Increases the ß-Carotene Content of the Endosperm.

Maize (Zea mays L.) is a staple food in many parts of Africa, but the endosperm generally contains low levels of the pro-vitamin A carotenoid ß-carotene, leading to vitamin A deficiency disease in populations relying on cereal-based diets. However, maize endosperm does accumulate high levels of other carotenoids, including zeaxanthin, which is derived from ß-carotene via two hydroxylation reactions. Blocking these reactions could therefore improve the endosperm ß-carotene content. Accordingly, we used RNA interference (RNAi) to silence the endogenous ZmBCH1 and ZmBCH2 genes, which encode two non-heme di-iron carotenoid ß-hydroxylases. The genes were silenced in a range of maize genetic backgrounds by introgressing the RNAi cassette, allowing us to determine the impact of ZmBCH1/ZmBCH2 silencing in diverse hybrids. The ß-carotene content of the endosperm increased substantially in all hybrids in which ZmBCH2 was silenced, regardless of whether or not ZmBCH1 was silenced simultaneously. However, the ß-carotene content did not change significantly in C17 hybrids (M7 × C17 and M13 × C17) compared to C17 alone, because ZmBCH2 is already expressed at negligible levels in the C17 parent. Our data indicate that ZmBCH2 is primarily responsible for the conversion of ß-carotene to zeaxanthin in maize endosperm.

Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed.

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.

Provitamin A carotenoids from an engineered high-carotenoid maize are bioavailable and zeaxanthin does not compromise ß-carotene absorption in poultry.

High-carotenoid (HC) maize, a biofortified staple crop which accumulates ß-carotene, ß-cryptoxanthin, lutein and zeaxanthin, was used as a feed component in a chicken feeding trial to assess the bioavailability of provitamin A (PVA) carotenoids in the kernel matrix compared to the synthetic and natural color additives routinely used in the poultry industry. We found that the PVA carotenoids in HC maize were not metabolized in the same manner: ß-carotene was preferentially converted into retinol in the intestine whereas ß-cryptoxanthin accumulated in the liver. We also considered the effect of zeaxanthin on the absorption of PVA carotenoids because zeaxanthin is the major carotenoid component of HC maize. We found that chickens fed on diets with low levels of zeaxanthin accumulated higher levels of retinol in the liver, suggesting that zeaxanthin might interfere with the absorption of ß-carotene, although this observation was not statistically significant. Our results show that HC maize provides bioavailable carotenoids, including PVA carotenoids, and is suitable for use as a feed component.

The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools.

KEY MESSAGE: The AtOR gene enhances carotenoid levels in corn by promoting the formation of plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant. The cauliflower orange (or) gene mutation influences carotenoid accumulation in plants by promoting the transition of proplastids into chromoplasts, thus creating intracellular storage compartments that act as metabolic sink. We overexpressed the Arabidopsis OR gene under the control of the endosperm-specific wheat LMW glutenin promoter in a white corn variety that normally accumulates only trace amounts of carotenoids. The total endosperm carotenoid content in the best-performing AtOR transgenic corn line was 32-fold higher than wild-type controls (~25 µg/g DW at 30 days after pollination) but the principal carotenoids remained the same, suggesting that AtOR increases the abundance of existing carotenoids without changing the metabolic composition. We analyzed the expression of endogenous genes representing the carotenoid biosynthesis and MEP pathways, as well as the plastid fusion/translocation factor required for chromoplast formation, but only the DXS1 gene was upregulated in the transgenic corn plants. The line expressing AtOR at the highest level was crossed with four transgenic corn lines expressing different carotenogenic genes and accumulating different carotenoids. The introgression of AtOR increased the carotenoid content of the hybrids when there was a limited carotenoid pool in the parental line, but had no effect when carotenoids were already abundant in the parent. The AtOR gene therefore appears to enhance carotenoid levels by promoting the formation of carotenoid-sequestering plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant because high levels of carotenoids can induce the formation of carotenoid-sequestering plastoglobuli even in the absence of AtOR.