VITAMIN K2 MK7 ALL TRANS

Vitamin K2 (menaquinone, MK) is in essential lipid-soluble vitamin with vital roles an blood coagulation and bone metabolism. Chemically, die term vitamin K2 encompasses a kopieren, gruppe of klein molecules that contain a usual naphthoquinone head group and a polyisoprenyl side chain of variable length. Among them, menaquinone-7 (MK-7) is the most potent form. Here, ns biosynthetic pathways von vitamin K2 und different types des MK produced über microorganisms are briefly introduced. Further, we provide a neu aspect des MK-7 production, which shares a usual naphthoquinone ring und polyisoprene biosynthesis pathway, by analyzing strategies zum expanding ns product spectrum. Us review ns findings von metabolic design strategies targeting die shikimate pathway, polyisoprene pathway, und menaquinone pathway, and membrane engineering, which administer comprehensive insights weil das enhancing the yield of MK-7. Finally, die current limitations and perspectives of microbial menaquinone manufacturing are also discussed. This post provides in-depth info on metabolic engineering strategies zum vitamin K2 production über expanding ns product spectrum.

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Introduction


Dr. Henrik Dam discovered vitamin K an the 1930s und shared die 1943 Nobel Prize bei medicine v Edward Doisy weil das their arbeiten on this fat-soluble bioactive link <1,2,3>. Vitamin K serves together a cofactor for γ-glutamyl carboxylase (GGCX), i beg your pardon converts glutamic acid residues of vitamin K-dependent proteins (VKDPs) right into γ-carboxyglutamic acid (Gla). Over there are more than fünfzehn types of VKDPs, consisting of coagulation factors ii (prothrombin), VII, IX, und X, as well as ns anti-coagulation determinants C, protein S, und osteocalcin <4,5,6,7>. Additionally, optimal vitamin K status is strongly associated with various health and wellness benefits, such as preventing or alleviating cardiovascular disease, osteoporosis, osteoarthritis, cancer, inflammation diseases, diabetes, chronic kidney disease, immune disorders, and Alzheimer’s an illness <4, 7,8,9,10>. Perhaps unsurprisingly, vitamin K deficiency may deshalb influence ns mortality und morbidity of covid-19 patients <11>.

The ax vitamin K denotes a series des fat-soluble compounds that contain a 2-methyl-1,4-naphthoqumone moiety together the basic skeleton and in isoprenoid chain at ns 3-position <12>. Based on the side-chain structure, vitamin K isoforms space categorized together vitamin K1 (phylloquinone, PK), vitamin K2 (menaquinone-n, MK-n) und vitamin K3 (menadione, MD) (Fig. 1) <13,14,15>. Vitamin K3 ist a artificial product there is no a next chain, if vitamin K1 und K2 happen naturally. Vitamin K1 has in aliphatic next chain and is predominately found in various leafy environment-friendly vegetables, fruits, and plant oil <13, 16>. Vitamin K2 contains in unsaturated aliphatic side chain v a variable number (n) von 4 zu 13 isoprene units and is referred kommen sie as MK-n <17>. Vitamin K1 zu sein currently produced via chemical synthesis, and there is little evidence des successful attempts zu reduce or eliminate ns utilization of toxic chemicals bei this process von applying enzyme <13, 16>. However, consumer are progressively favoring “natural” assets like vitamin K2 fermented by Generally recognized as for sure (GRAS) bacteria <18, 19>. Thus, this review focuses on strategies zum industrial vitamin K2 biosynthesis.


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The biosynthesis pathways von menaquinone an isoforms indigenous Bacillus subtilis, Saccharomyces cerevisiae und S. Coelicolor <26, 37, 41>. Menaquinone biosynthesis ist a complicated process involving multiple metabolic pathways, such together glycolysis, ns pentose phosphate pathway, ns shikimate pathway, die MEP or MVA pathway, as well as the classical MK pathway or futalosine pathway. a ns important intermediates chorismate und polyisoprene are synthesized from ns precursors G3P, pyruvate und E4P. b die classical pathway and the futalosine pathway weil das the biosynthesis von menaquinone. Enzymes room displayed an different colors bei different pathways. Red typeface suggests several vital intermediate metabolites


Among vitamin K2 homologues, MK-4 is the most common form bei animals, with ns widest range of physiological tasks <6>. The long-chain MK isoforms, such as MK-7, are found an fermented foods or produced von bacteria. The traditional Japanese food natto, which zu sein made from soybeans fermented über Bacillus subtilis natto, consists of MK-7 hinweisen a very high concentration <1>. MK-4 und MK-7 are allowed bei the united States as nutritional supplements zum bone health <20>. However, ns administration von MK-4 zu sein not reflected in an increased serum concentration <6>. Von contrast, MK-7 zu sein absorbed efficiently, which ist reflected in increased serum MK-7 level up kommen sie several days, thereby contributing zu vitamin K status <6, 21>. Us Pharmacopeia monographs oase been developed to establish quality standards zum menaquinone-7 as a diet ingredient punkt typically recommended level <22>. Although, there space cis, trans, und cis/trans isomers of MK-7, only the all-trans form is biologically energetic <21>. It can be created naturally v fermentation, as bei the case des natto, or von organic synthesis. In the process des organic synthesis des MK-7, cis/trans isomers kann be produced at the same time. Thus, the potential for the various isomeric forms von MK-7 to schutz different cytotoxic or giftig properties is an important consideration <21, 23>. Naturally acquired MK-7 zu sein produced über fermentation des generally known as safe (GRAS) bacterial strains such together B. Subtilis natto, und is therefore considered kommen sie be nontoxic <2>. Thus, enhancing ns production von vitamin K2 von environmentally trusted fermentation has actually been generally studied.

The aim of this review zu sein to offer neu insights zum improving microbial factories zum the production des vitamin K2, v a specific focus ~ above pathway engineering and membrane engineering von expanding the product spectrum. The bacterial MK biosynthesis pathway and the producing microorganism that synthesize different dominant MK isoforms (MK-4, MK-6, MK-7, MK-8, etc.) are briefly discussed. Finally, die limitations von microbial menaquinone production are also discussed.

Biosynthetic pathway des vitamin K2

The selection des high-yield strains zu sein based on die biosynthesis pathway of vitamin K2 an prokaryotes. In 1982, Bentley et al. <24> initially clarified die mechanism des vitamin K2 biosynthesis an bacteria and reviewed the detailed roles of shikimate, 2-succinylbenzoate, 1,4-dihydroxy-2-naphthoate, und other intermediates. They also described separation, personal, instance reactions an the pathway from ns perspective des chemical mechanisms und enzymological qualities <24>. Ns chemical structure des vitamin K2 has a naphthoquinone runde and an isoprene next chain <25>.

The naphthoquinone ring kann be synthesized via the classical MK pathway or the futalosine pathway (Fig. 1) <26,27,28>. The classical MK pathway has actually been known zum decades und is found in almost all aerobic or facultatively anaerobic prokaryotes. über contrast, ns futalosine-dependent pathway was zuerst discovered und validated an S. Coelicolor A3(2) in 2008, and is found an a more comprehensive taxonomic range des organisms, including also anaerobic microbe <29,30,31>. Both ns classical MK pathway und the futalosine pathway diverge hinweisen chorismate and re-converge after the gebildet of menaquinone <32>. Therefore, chorismate ist the key intermediate weil das the biosynthesis des the menaquinone ring. Chorismate zu sein synthesized via the shikimate pathway from two precursors: E4P, i beg your pardon is bei intermediate product of the pentose phosphate pathway, und phosphoenolpyruvate (PEP), in intermediate product des the glycolysis pathway or glycerol metabolism (Fig. 1a) <33, 34>. In the erste step von the classic MK pathway, MenF switch chorismate right into isochorismate, which is further converted von six enzymes encoded von the menDHCEB operon to form 1,4-dihydroxy-2-naphthoate (Fig. 1b) <17, 26>. In the zuerst step des the futalosine pathway, MqnA catalyzes die dehydration von chorismate kommen sie generate 3-<(1-carboxyvinyl) oxy>-benzoic acid, which is then converted right into 1,4-dihydroxy-6-naphthoate durch three enzymatic reactions catalyzed von MqnBCD (Fig. 1b) <31, 35, 36>.

Secondly, die isoprene next chain ist generally developed from two basic five-carbon units, isopentenyl diphosphate (IPP) und dimethylallyl diphosphate (DMAPP), either via die 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway or ns mevalonate (MVA) pathway <37, 38>. Ns MVA pathway ist present in most eukaryotes, archaea, part bacteria, as well as cytosol und mitochondria von plants, while most bacteria, chloroplasts, und apicomplexan parasites synthesize IPP and DMAPP through ns MEP pathway <37, 39>. Ns MEP pathway consists von eight enzyme-catalyzed reactions involved an the gebildet of IPP und DMAPP. Glyceraldehyde 3-phosphate (G3P) and pyruvate from die glycolysis pathway or glycerol metabolism space converted von 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) to form 1-deoxy-d-xylulose-5-phosphate (DXP), which enters the MEP pathway. Then, DXP kann be converted into MEP von 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr). IPP and DMAPP space produced über a pathway containing 5 enzymes, referred to as IspDEFGH, yet DMAPP can also be converted right into IPP by isopentenyl-diphosphate delta-isomerase (Fni) (Fig. 1a) <40>. Ns MEP pathway starts with die condensation des G3P and pyruvate, while the MVA pathway supplies acetyl‐CoA together a substrate. Acetyl-CoA zu sein converted right into acetoacetyl-CoA von acetyl-CoA acetyltransferase <41>. Then, the five enzyme Erg13, Hmg1/Hmg2, Erg12, Erg8, und Erg19 transform acetoacetyl-CoA to IPP. Finally, IPP undergoes a two-step catalytic reaction to form the isoprene next chain-polyprenyl-PP (C40) <41>. These two pathways schutz been extensively used in studies des isoprene biosynthesis.

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Finally, die membrane-bound enzyme polyprenyltransferase (MenA) and methyltransferase (MenG) combine the menaquinone ring and isoprene next chain zu synthesize vitamin K2 <40>.

Different isoforms des vitamin K2 produced von various bacteria

In the Western diet, cheese und curd created with Lactococcus lactis are ns most vital sources des vitamin K2. Bøe and Holo <42> co-overexpressed mvk, preA, und menA kommen sie increase ns vitamin K2 yield (mainly MK-3, MK-7, and MK-9) (Table 1). When milch was fermented using the modified K2-overproducing strains, ns vitamin K2 yield was effectively raised threefold compared zu the grausam type, which offers a foundation weil das the development von strains to gebrannt food with boosted functional worth <42>.


MK-4 ist found in small amounts an animal products, such together eggs and meat, and it is also the major form (> 90%) of vitamin K found in animal tissues <9, 43>. MK-4 is mainly produced von microbial fermentation, which only produces ns all-trans configuration, while die chemical synthesis von MK-4 remains a challeng <23>. Yuan et al. <44> improved the synthesis efficiency des MK-4 by combinatorial pathway design involving 4 modules, a MK-4 biosynthesis module (overexpression des menA, menG, und crtE), MEP module (knockout von hepT; overexpression of dxs, dxr, and ispD-ispF), MVA isoprenoid module (heterogeneous expression von mvaK1, mvaK2, mvaD, mvaS, and mvaA), and menaquinone module (overexpression of menA und menG). The MK-4 von resulting engineered B. Subtilis increased zu 120.1 mg/L in shake flasks (Table 1) <44>. An additional example is bei the methylotrophic yeast Pichia pastoris, bei which a novel synthetic pathway zum the production of MK-4 was introduced via heterologous expression des UbiA prenyltransferase include 1 (HsUBIAD1). After optimizing die expression conditions, die yield increased 4.37 zeit compared v that under the anfangsverdacht conditions (Table 1) <45>.

Escherichia coli zu sein a facultatively anaerobic bacterium the produces benzoquinone-type ubiquinone-8 (Q-8) under aerobic conditions and mainly synthesizes naphthoquinone-type menaquinone-8 (MK-8) under anaerobic conditions. Kong and Lee <46> enhanced the MK-8 content über modulating two precursors pools und blocking die competing synthesis of Q-8. Further, overexpression von menA and menD increased ns MK-8 productivity fivefold contrasted with die wild-type (Table 1) <46>.

Some enterobacteria such as Eubacterium lentum, Veillonella, Enterobacterium, und Bacteroides species kann sein produce MK-6, MK7, MK-8, MK-10, und MK-11, but ns exact mechanisms stay underexplored <47, 48>.

The MK-7 long-chain isoform is in essential nutrient for humans since it ist the most efficiently absorbed form of vitamin K and has a lang half-life von 68 h contrasted with just 1–2 h zum K1 <6, 49, 50>. B. Subtilis natto ist the main host zum the commercial production of vitamin K2 <51>. It was so shown kommen sie produce a range von vitamin K2 isoforms, indigenous MK-4 to MK-8, but MK-7 accounts zum more 보다 90% von the total <52>. B. Subtilis natto was originally used bei the fermentation von natto, a timeless Japanese food, i beg your pardon is an excellent source von MK-7 <53, 54>. Optimization des B. Subtilis natto expansion parameters including temperature, pH, and agitation an a glycerol-based tool increased ns MK-7 concentration kommen sie 12.09 mg/L in a biofilm reactor, without genetic alteration <55>. Next, die review möchte focus on ns metabolic engineering of microbes zum the production of MK-7.

Metabolic engineering of MK-7 manufacturing from ns aspects of pathways and the product spectrum

In ns pathways explained above, the most essential intermediates zum vitamin K2 biosynthesis space chorismate und isoprene. Chorismate enters the classical MK pathway or the futalosine pathway to form the naphthoquinone head-group (Fig. 1) <40>. Die polyisoprene tail ist produced indigenous two, five-carbon (C5) global precursors: IPP and DMAPP through the MEP pathway or ns MVA pathway <41>. Then, die polyisoprene tail ist ligated with ns naphthoquinone head by MenA to form demethylmenaquinone (DMK) and MK-7 zu sein synthesized via ns methylation of DMK <40>. Therefore, die key points des the pathways which might be die rate-limiting steps von vitamin K2 biosynthesis need to be discussed an depth. Undoubtedly, precursors zum vitamin K2 biosynthesis deshalb participate an the biosynthesis von various other chemicals, consisting of some well-studied ones, such together terpenoids und aromatic acids. Therefore, this review notfall only discusses ns unique metabolic engineering strategies zum the production von vitamin K2 but so offers a perspective des engineering methods zum other assets that share ns same precursors or pathways, thereby giving ideas zum future research on vitamin K2.

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Metabolic engineering von the shikimate pathway zu improve chorismate production

The shikimate pathway connects ns central carbon line with the biosynthesis of chorismate, which is a an essential precursor zum the production des aromatic amino acids und a huge number des other aromatic compounds in plants und microorganisms, including vitamin K2 (Fig. 2) <56,57,58>. In E. Coli, three different 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases (DAHPS) isoenzymes encoded von the AroGFH genes add to the total DAHPS activity and are subject to allosteric control von l-phenylalanine, l-tyrosine, and l-tryptophan, respectively <59,60,61,62>. Through structural analysis des mutant enzymes that are notfall sensitive to feedback, certain certain amino acid residues involved in the allosteric site schutz been identified, und feedback resistant (fbr) variants of AroG und AroF have been occurred <59, 62>. Hence, removed of feedback inhibition of the crucial enzymes obtained von introducing site-directed mutations ist usually the first and most vital step weil das the construction von a high-producing strain.