Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives.

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

A potential biotechnological process for the sustainable production of vitamin K1.

The primary objective of this review is to propose an approach for the biosynthesis of phylloquinone (vitamin K1) based upon its known sources, its role in photosynthesis and its biosynthetic pathway. The chemistry, health benefits, market, and industrial production of vitamin K are also summarized. Vitamin K compounds (K vitamers) are required for the normal function of at least 15 proteins involved in diverse physiological processes such as coagulation, tissue mineralization, inflammation, and neuroprotection. Vitamin K is essential for the prevention of Vitamin K Deficiency Bleeding (VKDB), especially in neonates. Increased vitamin K intake may also reduce the severity and/or risk of bone fracture, arterial calcification, inflammatory diseases, and cognitive decline. Consumers are increasingly favoring natural food and therapeutic products. However, the bulk of vitamin K products employed for both human and animal use are chemically synthesized. Biosynthesis of the menaquinones (vitamin K2) has been extensively researched. However, published research on the biotechnological production of phylloquinone is restricted to a handful of available articles and patents. We have found that microalgae are more suitable than plant cell cultures for the biosynthesis of phylloquinone. Many algae are richer in vitamin K1 than terrestrial plants, and algal cells are easier to manipulate. Vitamin K1 can be efficiently recovered from the biomass using supercritical carbon dioxide extraction.

A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis.

Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 Å resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 Å. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry.

Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

Molecular basis of the general base catalysis of an α/ß-hydrolase catalytic triad.

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/ß-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/ß-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.

Menaquinones, bacteria, and the food supply: the relevance of dairy and fermented food products to vitamin K requirements.

Vitamin K exists in the food supply as phylloquinone, a plant-based form and as menaquinones (MKs), a collection of isoprenologues mostly originating from bacterial synthesis. Although multiple bacterial species used as starter cultures for food fermentations synthesize MK, relatively little is known about the presence and distribution of MK in the food supply and the relative contribution of MK to total dietary vitamin K intake. Dairy products may be a predominant source of dietary MK in many regions of the world, and there is recent interest in enhancing the MK content of dairy products through identification and selection of MK-producing bacteria in dairy fermentations. This interest is increased by emerging evidence that current dietary recommendations based on the classic role of vitamin K as an enzyme cofactor for coagulation proteins may not be optimal for supporting vitamin K requirements in extrahepatic tissues and that MK may have unique bioactivity beyond that as an enzyme cofactor. Observational studies have reported favorable associations between MK intake and bone and cardiovascular health. Although randomized trials have provided some evidence to support the beneficial effects of MK on bone, the evidence to date is not definitive, and randomized trials have not yet examined MK intake in relation to cardiovascular outcomes. Food production practices provide a means to enhance dietary MK availability and intake. However, parallel research is needed to optimize these production practices, develop comprehensive food MK content databases, and test hypotheses of unique beneficial physiological roles of MK beyond that achieved by phylloquinone.

Bacteria as vitamin suppliers to their host: a gut microbiota perspective.

Food-related lactic acid bacteria (LAB) as well as human gut commensals such as bifidobacteria can de novo synthesize and supply vitamins. This is important since humans lack the biosynthetic capacity for most vitamins and these must thus be provided exogenously. Although vitamins are present in a variety of foods, deficiencies still occur, mainly due to malnutrition as a result of insufficient food intake and because of poor eating habits. Fermented milks with high levels of B-group vitamins (such as folate and riboflavin) can be produced by LAB-promoted and possibly bifidobacteria-promoted biosynthesis. Moreover, certain strains of LAB produce the complex vitamin cobalamin (or vitamin B12). In this review, fermented foods with elevated levels of B-group vitamins produced by LAB used as starter cultures will be covered. In addition, genetic abilities for vitamin biosynthesis by selected human gut commensals will be discussed.

Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes.

Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as ß-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann-Pick C1-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular mechanisms involved in fat-soluble vitamin and carotenoid absorption is a priority to better optimize their bioavailability.

Metabolic engineering of menaquinone-8 pathway of Escherichia coli as a microbial platform for vitamin K production.

Menaquinone-8 (MK-8, vitamin K) is composed of a non-polar side chain and a polar head group. Escherichia coli was chosen and metabolically engineered as a microbial platform for production of MK-8. MK-8 content in E. coli was significantly enhanced by modulating two precursor pools, which supply a non-polar side chain and a polar head group, and further increased by blocking formation of the competitor ubiquinone-8 (Q-8). Overexpression of E. coli IspA, DXR, or IDI increased MK-8 content up to twofold. A similar positive effect was also observed when E. coli MenA, MenB, MenC, MenD, MenE, MenF, or UbiE was overexpressed. The Q-8-deficient ubiCA mutant enhanced MK-8 content by 30% compared to wild-type E. coli. When MenA or MenD was overexpressed, MK-8 content was enhanced fivefold compared with wild-type E. coli.

Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

OBJECTIVE: Mineralization has been observed in osteoarthritic cartilage but the mechanisms are incompletely understood. Vitamin K is an essential cofactor in post-translational modification of proteins where specific Glu residues become modified to Ca(++) binding gamma-carboxyglutamic acid residues (Gla). One such protein, matrix Gla protein (MGP), is a known mineralization inhibitor. This study determined if synthesis of MGP and formation of a fetuin-MGP protein complex was altered in chondrocytes and vesicles from osteoarthritis (OA) cartilage. METHODS: Chondrocytes and vesicles were isolated from normal and OA human articular cartilage and lysates prepared. Specific antibodies were used in immunoblotting to detect the mature fully gamma-carboxylated form of MGP (cMGP) and non-gamma-carboxylated MGP (ucMGP) as well as fetuin and MGP-fetuin complexes. gamma-carboxylase activity was measured by (14)CO(2) incorporation into the carboxylase peptide substrate FLEEL. Immunocytochemistry was used to examine fetuin in cartilage sections and uptake of biotin-labeled fetuin by isolated chondrocytes. RESULTS: Chondrocytes and vesicles from osteoarthritic tissue produced significantly less cMGP compared to those from normal cartilage. This correlated with significantly less vitamin K-dependent gamma-carboxylase enzyme activity in OA chondrocytes. Fetuin was found to be present in articular cartilage and cultured chondrocytes were capable of fetuin uptake. A fetuin-MGP complex was identified in normal chondrocytes and in vesicles shed from these cells but not in OA cells or vesicles. CONCLUSIONS: The absence of cMGP and of the cMGP-fetuin complex in OA cells and OA vesicles may be an important mechanism for increased mineralization of osteoarthritic cartilage.

Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2.

More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is approximately 21 kDa, as expected; fully active; and > 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

Warfarin as a therapeutic option in the control of chronic cluster headache: a report of three cases.

Chronic cluster headache remains refractory to medical therapy in at least 30% of those who suffer from this condition. The lack of alternative medical therapies that are as effective as, or more effective than, lithium carbonate makes new therapies necessary for this highly disabling condition. Based on a previous report, we gave oral anticoagulants to three patients with chronic cluster headache. Two of them remained cluster headache-free while taking warfarin. In the third patient, the use of warfarin for three weeks initially increased the frequency and intensity of cluster headache attacks but subsequently induced a prolonged remission. In spite of the paucity of data available, oral anticoagulation appears to be a promising therapy for chronic cluster headache.

Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis.

Efficient production of menaquinone (MK) by Bacillus subtilis was achieved. An edible strain of B. subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity. A menadione-resistant mutant producing 30% more MK than its parent strain was obtained. Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested. Addition of yeast extract also increased MK productivity. The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter. The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7-8.0. This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0.

Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms.

The benzoquinone ubiquinone (coenzyme Q) and the naphthoquinones menaquinone (vitamin K2) and demethylmenaquinone are derived from the shikimate pathway, which has been described as a "metabolic tree with many branches." Menaquinone (MK) is considered a vitamin, but coenzyme (Q) is not; MK is an essential nutrient (it cannot be synthesized by mammals), whereas Q is not considered an essential nutrient since it can be synthesized from the amino acid tyrosine. The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is derived from chorismate via isochorismate. The prenyl side chain of both quinones is derived from prenyl diphosphate, and the methyl groups are derived from S-adenosylmethionine. MK biosynthesis requires 2-ketoglutarate and the cofactors ATP, coenzyme A (CoASH), and thiamine pyrophosphate. In spite of the fact that both quinones originate from the shikimate pathway, there are important differences in their biosynthesis. In MK biosynthesis, the prenyl side chain is introduced in the next to last step, which is accompanied by loss of the carboxyl group, whereas in Q biosynthesis, the prenyl side chain is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to the prenylation (next to last step) are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. In spite of the fact that Q and MK biosynthesis diverges at chorismate, the C-methylations involved in both pathways are carried out by the same enzyme. Finally, Q biosynthesis under aerobic conditions requires molecular oxygen; anaerobic biosynthesis of Q and MK incorporates oxygen atoms derived from water. The current status of the pathways with particular emphasis on the reaction mechanisms, is discussed in this review.

A role for excreted quinones in extracellular electron transfer.

Respiratory processes in bacteria are remarkable because of their ability to use a variety of compounds, including insoluble minerals, as terminal electron acceptors. Although much is known about microbial electron transport to soluble electron acceptors, little is understood about electron transport to insoluble compounds such as ferric oxides. In anaerobic environments, humic substances can serve as electron acceptors and also as electron shuttles to ferric oxides. To explore this process, we identified mutants in Shewanella putrefaciens that are unable to respire on humic substances. Here we show that these mutants contain disruptions in a gene that is involved in the biosynthesis of menaquinone. During growth, the wild type releases a menaquinone-related redox-active small molecule into the medium that complements the mutants. This finding raises the possibility that electron transfer to a variety of oxidants, including poorly soluble minerals, may be mediated by microbially excreted quinones that have yet to be identified.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Production of menaquinones by lactic acid bacteria.

Lactic acid bacteria were examined for their ability to produce quinone compounds, which may include dietary sources of menaquinones. Isoprenyl quinones in bacterial cells grown in a synthetic medium were extracted and analyzed by thin layer chromatography. Lactococcus lactis ssp. cremoris (three strains), Lactococcus lactis ssp. lactis (two strains), and Leuconostoc lactis were selected as high producers of quinone that synthesized more than 230 nmol of quinones/g of dried cells. The quinones were presumed to be menaquinone-7 to -10 by high performance liquid chromatography. Precise molecular weights were determined by mass spectrometry for Lactococcus lactis ssp. cremoris YIT 2011 and Leuconostoc lactis YIT 3001 and identified as menaquinone-8 and -9 for the former and menaquinone-9 and -10 for the latter. Those strains, when grown either in reconstituted nonfat dry milk or a soymilk medium, produced a beneficial quantity for dietary supplement (i.e., 29 to 123 micrograms of menaquinones/L of the fermented medium).

The role of isochorismate hydroxymutase genes entC and menF in enterobactin and menaquinone biosynthesis in Escherichia coli.

Klebsiella pneumoniae 62-1, a triple mutant impaired in aromatic amino acid biosynthesis (Phe-, Tyr-, Trp-), excretes chorismic acid into the culture broth. When transformed with plasmids harbouring Escherichia coli genes entC or menF the mutant excretes a mixture of both chorismic and isochorismic acid indicating that not only entC but also menF encodes an isochorismate hydroxymutase (isochorismate synthase, EC 5.4.99.6) enzyme. These enzymes catalyze the first step in enterobactin or menaquinone biosynthesis, respectively. Although both gene products (EntC and MenF) catalyze the same reaction, they play distinct roles in the biosynthesis of menaquinone (MK8) and enterobactin. An E. coli mutant (PBB7) with an intact menF but a disrupted entC produced menaquinone (MK8) but no enterobactin, whereas a mutant (PBB9) with an intact entC but a disrupted menF produced enterobactin and only a trace of menaquinone (MK8). When both menF and entC were disrupted (mutant PBB8) neither menaquinone (MK8) nor enterobactin was detectable. Our previous assumption that entC is responsible for both menaquinone and enterobactin biosynthesis is inconsistent with these mutant studies and has to be revised. The presence in the promoter region of menF of a putative cAMP receptor protein binding site indicates that menF is regulated differently from entC. The menF gene was overexpressed as a fusion gene and its product (6xHis-tagged MenF) isolated. The enzyme catalyzed the formation of isochorismic from chorismic acid and as opposed to a previous publication also the reverse reaction. The enzyme was characterized and its kinetic data determined.

Interaction between vitamin K nutriture and bacterial overgrowth in hypochlorhydria induced by omeprazole.

Subjects taking a hydrogen pump blocking agent (omeprazole) develop bacterial overgrowth of the small intestine. We tested the hypothesis that this bacterial overgrowth produces menaquinones, which would meet the vitamin requirement in situations of vitamin K deficiency. In a crossover-type design, 13 healthy volunteers eating a phylloquinone-restricted diet for 35 d were randomly assigned to take omeprazole during the first period of study or starting on day 15 until the end of the study. Coagulation times, serum osteocalcin [total osteocalcin and undercarboxylated osteocalcin (ucOC)], plasma phylloquinone, urinary gamma-carboxyglutamic acid, and plasma undercarboxylated prothrombin (PIVKA-II) were measured. Plasma phylloquinone concentrations declined 82% with dietary phylloquinone restriction (P < 0.05) and were not significantly different in the period when the diet was combined with omeprazole treatment (P > 0.05). The mean value for PIVKA-II during the phylloquinone-restricted diet significantly increased 5.7-fold from baseline (P < 0.05); however, the combination of omeprazole treatment and the phylloquinone-restricted diet significantly reduced PIVKA-II values by 21% (P < 0.05) compared with the diet period alone. There were no alterations in total or percentage ucOC concentrations during the phylloquinone-restricted diet or during the period of diet plus omeprazole treatment. Our data support the hypothesis that bacterial overgrowth results in the synthesis and absorption of menaquinones. These menaquinones contribute to vitamin K nutriture during dietary phylloquinone restriction, but not enough to restore normal vitamin K status.