vitamins

11 Vitamins and RelatedCompounds: Microbial Production

SAKAYU SHIMIZUKyoto, Japan

1 Introduction 3202 Water-Soluble Vitamins 320

2.1 Riboflavin (Vitamin B2) and Related Coenzymes 3202.2 Nicotinic Acid, Nicotinamide, and Related Coenzymes 3232.3 Pantothenic Acid and Coenzyme A 3252.4 Pyridoxine (Vitamin B6) 3272.5 Biotin 3282.6 Vitamin B12 3282.7 L-Ascorbic Acid (Vitamin C) 3282.8 Adenosine Triphosphate and Related Nucleotides 3302.9 S-Adenosylmethionine and Related Nucleosides 3312.10 Miscellaneous 331

3 Fat-Soluble Vitamins 3323.1 Vitamin A (Retinoids) and �-Carotene (Provitamin A) 3323.2 Vitamin D 3333.3 Tocopherols (Vitamin E) 3343.4 Polyunsaturated Fatty Acids (Vitamin F Group) 3353.5 Vitamin K Compounds 3353.6 Ubiquinone Q (Coenzyme Q) 336

4 Concluding Remarks 3365 References 337

320 11 Vitamins and Related Compounds: Microbial Production

1 Introduction

Vitamins are defined as essential micronu-trients that are not synthesized by mammals.Most vitamins are essential for the metabolismof all living organisms, and they are synthe-sized by microorganisms and plants. Coen-zymes (and/or prosthetic groups) are definedas organic compounds with low molecularweight that are required to show enzyme ac-tivity by binding with their apoenzymes. Manycoenzymes are biosynthesized from vitaminsand contain a nucleotide (or nucleoside)moiety in their molecules. Besides their func-tions as vitamins and coenzymes, most of vit-amins and coenzymes have been shown tohave various other biofunctions. Accordingly,it is more appropriate to understand both aseffective biofactors (see FRIEDRICH, 1988 forbasic information).

Most vitamins and related compounds arenow industrially produced and widely used asfood or feed additives, medical or therapeuticagents, health aids, cosmetic and technical aids,and so on. Thus, vitamins and related com-pounds are important products for whichmany biotechnological production processes(i.e., fermentation and microbial/enzymatictransformation) as well as organic chemicalsynthetic ones have been reported; some ofthem are now applied for large-scale produc-tion. Industrial production methodology, an-nual production amounts, and fields of appli-cation for these vitamins and related com-pounds are summarized in Tabs. 1–3.

In this chapter, some of the vitamins and re-lated compounds are described from the view-point of their microbial production. Previousreviews, from a similar viewpoint, may be use-ful for further information (DE BAETS et al.,2000; EGGERSDORFER et al., 1996; FLORENT,1986; SHIMIZU and YAMADA, 1986; VANDAM-ME, 1989).

2 Water-Soluble Vitamins

2.1 Riboflavin (Vitamin B2) andRelated Coenzymes

Riboflavin is used for human nutrition andtherapy and as an animal feed additive. Thecrude concentrated form is also used for feed.It is produced by both synthetic and fermenta-tion processes [major producers, Hoffmann-La Roche (Switzerland), BASF (Germany),ADM (USA), Takeda (Japan)]. The currentworld production of riboflavin is about 2,400 t aP1, of which 75% is for feed additiveand the remaining for food and pharmaceuti-cals. Two closely related ascomycete fungi,Eremothecium ashbyii and Ashbya gossypii,are mainly used for the industrial production(OZBAS and KUTSAL, 1986; STAHMANN et al.,2000). Yields much higher than 10 g of ribo-flavin per liter of culture broth are obtained ina sterile aerobic submerged fermentation witha nutrient medium containing molasses orplant oil as a major carbon source. Yeasts(Candida flaeri, C. famata, etc.) and bacteriacan also be used for the practical production.Riboflavin production by genetically engi-neered Bacillus subtilis and Corynebacteriumammoniagenes which overexpress genes of theenzymes involved in riboflavin biosynthesisreach 4.5 g LP1 and 17.4 g LP1, respectively(KOIZUMI et al., 1996; PERKINS et al., 1999).D-Ribose is used as the starting material in thechemical production processes, in which it istransformed to riboflavin in three steps.D-Ribose is obtained directly from glucose byfermentation with a genetically engineeredBacillus strain which is transketolase-de-fective and overexpresses the gluconateoperon (DE WULF and VANDAMME, 1997).

Flavin mononucleotide (FMN), a coenzymeform of riboflavin, is synthesized from ribo-flavin by chemical phosphorylation, afterwhich FMN is crystallized as the diethanol-amine salt to separate isomeric riboflavinphosphates and unreacted riboflavin.

The other coenzyme form of riboflavin, fla-vin adenine dinucleotide (FAD), is used inpharmaceutical and neutraceutical applicati-ons. Several tons of FAD are annually pro-

Tab. 1. Industrial Production of Vitamins and Coenzymes

Compound Production Method World Production Use[t aP1]

Biotechno- Chem- Extrac- 1980sa 1990sb

logical ical tion

Thiamin (B1) c 1,700 4,200 food, pharmaceuticalRiboflavin (B2) c 2,000 2,400 feed, pharmaceuticalFAD c c 10 pharmaceuticalNicotinic acid, c c 8,500 22,000 feed, food, pharmaceuticalnicotinamide

NAD, NADP c technicalPantothenic acid cc c 5,000 7,000 feed, food, pharmaceuticalCoenzyme A c technical, neutraceuticalPyridoxine (B6) c 1,600 2,550 feed, food, pharmaceuticalBiotin (c)d c 2.7 25 feed, pharmaceuticalFolic acid c 100 400 feed, food, pharmaceuticalVitamin B12 c 12 10 feed, food, pharmaceuticalVitamin C cc 40,000 60,000 feed, food, pharmaceuticalATP c pharmaceutical, technicalS-Adenosyl- c pharmaceutical, nutraceuticalmethionine

Lipoic acid c pharmaceuticalPyrroloquinoline c c technicalquinone

Vitamin A c 2,500 2,700 feed, food, pharmaceutical�-Carotene c c 100 400 feed, foodErgosterol c 25 38 feed, foodVitamin D3 c c 5,000 feed, food�-Tocopherol (E) (c)d c c 6,800 22,000 feed, food, pharmaceutical,

nutraceuticalPUFAse c c feed, food, pharmaceutical,

nutraceuticalPhylloquinone (K1) c 3.5 pharmaceuticalMenaquinone (K2) c 500 pharmaceuticalUbiquinone-10 c feed, food, pharmaceutical

a Values were taken from FLORENT (1986).b Values were taken from EGGERSDORFER et al. (1996).c Hybrid of microbial and chemical reactions.d Parentheses indicate pilot scale process.e PUFAs, polyunsaturated fatty acids.

2 Water-Soluble Vitamins 321

duced by chemical synthesis or by microbialtransformation. The latter uses FMN andadenosine 5b-triphosphate (ATP) as the sub-strates and C. ammoniagenes cells as a sourceof FMN adenylyltransferase. In this trans-formation,ATP is generated from adenine andphosphoribosyl pyrophosphate is de novo syn-thesized from glucose by the same organism

(see Sect. 2.8). In a similar fashion using the C. ammoniagenes ATP generating system, ge-netically engineered strains of Escherichia coliwhich overexpress flavokinase, and FMNadenylyltransferase can be used as the catalystin the transformation from riboflavin (KITA-TSUJI et al., 1992) (Fig. 1).


Tab. 2. Microbial and Enzymatic Processes for the Production of Water-Soluble Vitamins and Coenzymes

Vitamin, Coenzyme Enzyme (Microorganism) Method

Vitamin C 2,5-diketo-D-gulonic acid reductase enzymatic conversion of 2,5-(2-Keto-L-gulonic acid) (Corynebacterium sp.) diketo-D-gluconate obtained

through fermentative process to 2-keto-L-gulonic, followed by chemi-cal conversion to L-ascorbic acid

Biotin fermentation (Serratia marcescens) fermentative production from glu-cose by a genetically engineeredbacterium

multiple enzyme system conversion from diaminopimelic(Bacillus sphaericus) acid using the biotin biosynthesis

enzyme system of a mutant of B. sphaericus

Pantothenic acid lactonohydrolase resolution of D,L-pantolactone to(D-Pantoic acid) (Fusarium oxysporum) D-pantoic acid and L-pantolactone

by stereoselective hydrolysis

Coenzyme A multiple enzyme system conversion by enzymatic coupling(Brevibacterium ammoniagenes) of ATP-generating system and

coenzyme A biosynthesis systemof B. ammoniagenes (parent strainor mutant) with D-pantothenicacid, L-cysteine, and AMP (oradenosine, adenine, etc.) assubstrates

Nicotinamide nitrile hydratase hydration of 3-cyanopyridine(Rhodococcus rhodochrous)

Nicotinic acid nitrilase hydrolysis of 3-cyanopyridine to(Rhodococcus rhodochrous) form corresponding acid (nicotinic

acid) and ammonia

NAD multiple enzyme system conversion by enzymatic coupling (Corynebacterium ammoniagenes) of ATP-generating system and

NAD biosynthesis enzymes of B. ammoniagenes with adenineand nicotinamide as substrates

NADP NAD kinase (Brevibacterium sp., phosphorylation of NAD withCorynebacterium sp., etc.) ATP as the phosphate group

donor

NADH formic acid dehydrogenase reduction of NAD with formic (Arthrobacter sp., Candida boidinii, etc.) acid as the hydrogen donor

NADPH glucose dehydrogenase reduction of NADP with glucvose(Bacillus sp., Gluconobacter sp., etc.) as the hydrogen donor

Riboflavin fermentation (Eremothecium ashbyii, fermentative production fromAshbya gossypii, Bacillus sp., etc.) glucose


2.2 Nicotinic Acid, Nicotinamide,and Related Coenzymes

The world production of nicotinic acid andnicotinamide is estimated to be 22,000 t aP1

[major producers, BASF, Lonza (Switzerland)and Degussa (Germany)]. The major use (ca.75%) is for animal nutrition and the remainingfor food enrichment and pharmaceutical appli-cation. Chemical processes involving oxidationof 5-ethyl-2-methylpyridine or total hydrolysis

of 3-cyanopyridine are used for nicotinic acidproduction. Bacterial nitrilase has been shownto be useful for the same purpose (Fig. 2a). Forexample, 3-cyanopyridine is almost stoichio-metrically converted to nicotinic acid (172 g LP1) on incubation with the nitrilase-overexpressed Rhodococcus rhodochrous J1cells (NAGASAWA and YAMADA, 1989). Thesame R. rhodochrous enzyme can be used forthe production of p-aminobenzoic acid from p-aminobenzonitrile.

Tab. 2. Continued

Vitamin, Coenzyme Enzyme (Microorganism) Method

FAD FAD synthetase (Corynebacterium sp., enzymatic pyrophosphorylationArthrobacter sp., etc.) of ATP and flavin mononucleotide

synthesized chemically

ATP multiple enzyme system (baker’s yeast, ribotidization of adenine (or methylotrophic yeasts, Corynebacterium adenosine) under coupling of theammoniagenes, etc.) glycolysis system or methanol oxi-

dation system

S-Adenosylmethionine S-adenosylmethionine synthetase conversion of L-methionine by(Saccharomyces saké) S. saké mutant

S-Adenosylhomocysteine S-adenosylhomocysteine hydrolase condensation of adenosine and(Alcaligenes faecalis) homocysteine

L-Carnitine �-oxidation-like enzymes conversion of butyrobetaine to(Agrobacterium sp.) L-carnitinealdehyde reductase enzymatic asymmetric reduction(Sporobolomyces salmonicolor) of 4-chloroacetoacetic acid ester

to R-(P)-3-hydroxy-4-chloro-butanoic acid ester, followed by itschemical conversion to L-carnitine

Pyridoxal-5b-phosphate pyridoxamine oxidase oxidation of chemically(Pseudomonas sp.) synthesized pyridoxine-5b-phos-

phate

CDP-choline, CDP-choline pyrophosphorylase, pyrophosphoric acid condensationGDP-glucose, etc. NDP-glucose pyrophosphorylase, etc. of choline (or glucose, etc.) and

(yeasts, etc.) nucleotide triphosphate (or thecorresponding nucleoside)

Vitamin B12 fermentation fermentative production from(Propionibacterium shermanii, glucosePseudomonas denitrificans, etc.)

Pyrroloquinoline fermentation (methanol-utilizing fermentative production fromquinone (PQQ) bacterium) methanol

Nicotinamide is available from partial hy-drolysis of 3-cyanopyridine, which is perform-ed by both chemical and enzymatic processes.The enzymatic process uses nitrile hydrataseas the catalyst (Fig. 2b). This novel enzyme ca-talyzing a simple hydration reaction was dis-covered as one of the responsible enzymes forthe two-step transformation of nitriles to acidsvia amides (ASANO et al., 1980). Extensive stu-dies of this enzyme as well as screening of theenzyme from a variety of microbial strains re-vealed the presence of several different types


Tab. 3. Microbial and Enzymatic Processes for the Production of Fat-Soluble Vitamins

Vitamin Enzyme (Microorganism) Method

Vitamin E and K1 side chains multiple enzyme system enzymatic conversion from(Geotrichum candidum) (E)-3-(1b,3b-dioxolane-2b-yl)-2-butene-1-ol

[(S)-2-methyl-�-butyrolactone] reductase bakers’ yeast, asymmetric reduction of ethyl-4,4-[(S)-3-methyl-�-butyrolactone] (Geotrichum sp., etc.) dimethoxy-3-methylcrotonate[(S)- or (R)-�-hydroxy- multiple enzyme system stereoselective oxidation of isobutyric acidisobutyric acid] (Candida sp., etc.)

Vitamin K2 multiple enzyme system conversion of quinone- and side chain-(Flavobacterium sp.) precursors to the vitamin

Arachidonic acid fermentation fermentative production from glucose(Mortierella alpina)

Dihomo-�-linolenic acid fermentation fermentative production from glucose by (Mortierella alpina) a �5-desaturase-defective mutant

Mead acid fermentation fermentative production from glucose by(Mortierella alpina) a �12-desaturase-defective mutant

Eicosapentaenoic acid multiple enzyme system �17-desaturation of arachidonic acid or (Mortierella alpina) conversion from �-linolenic acid

Fig. 1. Schematic representation for the FAD pro-duction from riboflavin (RF) coupled with bacterialATP-generating system (see also Fig. 8).

Fig. 2. Transformation of 3-cyanopyridine to nicoti-nic acid by nitrilase a and nicotinamide by nitrile hy-dratase b.

of nitrile hydratases, especially Co- and Fe-containing enzymes, in various bacteria (KO-BAYASHI et al., in press).The Co-containing en-zyme from R. rhodochrous J1 hydrates variouskinds of aliphatic and aromatic nitriles to thecorresponding amides and has been shown tobe useful for the production of useful amides(YAMADA and KOBAYASHI, 1996). For example,using the bacterial cells containing highly ele-vated amounts of this enzyme exceeding 50%of the total cellular proteins, 1.23 kg of 3-cya-nopyridine suspended in 1 liter of water are


stoichiometrically converted to 1.46 kg of ni-cotinamide crystals (NAGASAWA and YAMADA,1989). Based on these studies, Lonza (Switzer-land) has constructed a plant for the commer-cial production of nicotinamide in China in1997. This enzymatic process surpasses thechemical process in regard to several pointssuch as stoichiometric conversion of high con-centration of the substrate and the quality ofthe product actually with zero contents of by-products. The same enzyme has been used forthe industrial production of acrylamide fromacrylonitrile by Nitto (Japan) since 1991.

Nicotinamide adenine dinucleotide (NAD)is used in pharmaceutical application and as areagent for clinical analysis. Nicotinamideadenine dinucleotide phosphate (NADP) isalso used for analysis. Practical production ofNAD is carried out by extraction. Yeasts suchas Saccharomyces cerevisiae are favorablesources of NAD. It is also produced by micro-bial transformation utilizing the salvage path-way for the biosynthesis of NAD from nico-tinamide (or nicotinic acid) and ATP. On culti-vation of Corynebacterium ammoniageneswith the precursors, nicotinamide and adenine,the amount of NAD in the medium reaches 2.3 mg mLP1 (NAKAYAMA et al., 1968). For themechanism involved in the transformation ithas been suggested that both precursors arefirst ribotidated to nicotinamide monophos-phate and ATP, respectively, which are thenconverted to NAD by pyrophosphorylation(for ATP generation, see Sect. 2.8). NADP canbe prepared by enzymatic phosphorylation.Reduced forms of these coenzymes, NADHand NADPH, can be obtained by both chem-ical and enzymatic methods. The latter usesformate dehydrogenase from methanol-utiliz-ing yeasts for NADH. Glucose dehydrogenasefrom Bacillus sp. is also used for both NADHand NADPH. In situ regeneration of thesecoenzymes is currently attracting more atten-tion for the production of chiral alcohols fromprochiral carbonyl compounds with carbonylreductases (Fig. 3). For example, geneticallyengineered E. coli cells overexpressing glucosedehydrogenase from Bacillus sp. and aldehydereductase from Sporobolomyces salmonicoloreffectively catalyze stereospecific reduction ofethyl 4-chloro-3-oxobutanoate to ethyl R(P)-4-chloro-3-hydroxybutanoate in the presence

of glucose and a catalytic amount of NADP(SHIMIZU and KATAOKA, 1999a; SHIMIZU et al.,1997).

2.3 Pantothenic Acid andCoenzyme A

About 6,000 t of calcium D-pantothenate areproduced annually. It is mainly used as an ani-mal feed additive (80%). It is also used forpharmaceutical, health care and food prod-ucts. D-Pantothenyl alcohol (1,000 t aP1) is alsoused for the same purposes. The commercialproduction process involves reactions yieldingracemic pantolactone from isobutyraldehyde,formaldehyde, and cyanide, optical resolutionof the racemic pantolactone to D-pantolac-tone, and condensation of D-pantolactone with�-alanine to form D-pantothenic acid. 3-Ami-nopropanol is used for D-pantothenyl alcohol[major producers, Hoffmann-La Roche, Fuji(Japan), and BASF]. The conventional opticalresolution which requires expensive alkaloidsas resolving agents is troublesome. Recently,an efficient enzymatic method has been intro-duced into this optical resolution step (SHIMI-ZU et al., 1997). This enzymatic resolution usesa novel fungal enzyme, lactonohydrolase, asthe catalyst. The enzyme catalyzes stereospe-cific hydrolysis of various kinds of lactones.D-Pantolactone is a favorable substrate of thisenzyme, but the L-enantiomer is not hydrolyzed

Fig. 3. In situ NAD(P)H regeneration with glucose de-hydrogenase (GDH) for the stereospecific reductionof prochiral carbonyl compounds to chiral alcohols.

at all (SHIMIZU et al., 1992). Thus, the racemicmixture can be separated into D-pantoic acidand L-pantolactone (Fig. 4). As this lactonasereaction is an intermolecular ester bond hy-drolysis, the pantolactone as the substrateneeds not to be modified for resolution, whichis one of the practical advantages of the use ofthis enzyme. Several filamentous fungi of thegenera Fusarium, Gibberella, and Cylindro-carpon show high activity of this enzyme. Onincubation with Fusarium oxysporum cells for24 h at pH 7.0, D-pantolactone in a racemicmixture (700 g LP1) is almost completely hy-drolyzed to D-pantoic acid (96% ee) (KATAO-KA et al., 1995a, b). Practically, this stereospe-cific hydrolysis is carried out by F. oxysporumcells immobilized with calcium alginate gels.When the immobilized cells were incubated ina racemic mixture (350 g LP1) for 21 h at 30 °C,90–95% of the D-pantolactone was hydrolyzedto D-pantoic acid (90–97% ee). After repeated

reaction for 180 times (i.e., 180 d), the immobi-lized cells retained about 90% of their initialactivity (SHIMIZU and KATAOKA, 1996, 1999b;SHIMIZU et al., 1997). The overall process forthis enzymatic resolution is compared with theconventional chemical process in Fig. 5. Theenzymatic process can skip several tedioussteps which are necessary in the chemical reso-lution. Based on these studies, Fuji (Japan)changed over their chemical resolution withthis enzymatic resolution in 1999.

Several enzymatic methods to skip this reso-lution step have also been reported. The two-step chemicoenzymatic method, which in-volves a one-pot synthesis of ketopantolactoneand its stereospecific reduction to D-pantolac-tone (SHIMIZU and YAMADA, 1989b; SHIMIZU

et al., 1997) is practically promising.The chem-ical synthesis is performed in one step fromisobutyraldehyde, sodium methoxide, diethyloxalate, and formalin at room temperature


Fig. 4. Principle of the optical resolutionof D,L-pantolactone by fungal lactonase.PL, pantolactone; PA, pantoic acid.

Fig. 5. Comparison of chemical and enzymatic methods for the optical resolution of D,L-pantolactone.For abbreviations, see Fig. 4.


with a yield of 81%. The bioreduction is per-formed in the presence of glucose as an energysource for the reduction and Candida parapsi-losis cells with high carbonyl reductase activityas the catalyst. In this bioreduction, ketopanto-lactone is stoichiometrically converted to D-pantolactone (90 g LP1, 94% ee) with a molaryield of 100% (HATA et al., 1987). Alternative-ly, ketopantoic acid, which is easily obtainedfrom ketopantolactone by spontaneous hydro-lysis under mild alkaline conditions, can beused as the substrate for the stereospecific bio-reduction. In this case, Agrobacterium sp. cellswith high activity of ketopantoic acid reduct-ase are used as the catalyst. The yield of D-pantoic acid was 119 g LP1 (molar yield,90%; optical purity, 98% ee) (KATAOKA et al.,1990).The chemical step can be replaced by anenzymatic one using L-pantolactone dehydro-genase of Nocardia asteroides. The enzymespecifically oxidizes the L-isomer in a racemicpantolactone mixture to ketopantolactone,which is then converted to D-pantolactone orD-pantoic acid by the above mentioned reduc-tion with C. parapsilosis or Agrobacterium sp.,respectively (KATAOKA et al., 1991a, b; SHIMI-ZU et al., 1987) (Fig. 6).

A direct fermentation process for D-pantoicacid and/or D-pantothenic acid is also promis-ing. A genetically engineered strain of E. colioverexpressing pantothenic acid biosynthesisenzymes produced 65 g LP1 D-pantothenicacid from glucose upon addition of �-alanineas a precursor (HIKICHI et al., 1993).

Coenzyme A (CoA) is used as an analyticalreagent and for pharmaceutical, neutraceuti-cal, and cosmetic applications. A successfulmicrobial transformation method uses Brevi-bacterium ammoniagenes cells, in which all fiveenzymes necessary for the biosynthesis ofCoA from D-pantothenic acid, L-cysteine, and

ATP abundantly occur, as the catalyst, andthese three precursors as the substrates (SHI-MIZU and YAMADA, 1986, 1989b). On cultiva-tion of the bacterium in a medium containingglucose (10%), D-pantothenic acid, L-cysteineand AMP (or adenine), from which ATP is ef-fectively generated by the same bacterium(see Sect. 2.8), the yield of CoA was 3–6 g LP1.Higher yields (ca. 20 g LP1) were obtainedwhen 4b-phosphopantothenic acid was used inplace of D-pantothenic acid or oxypantetheine-resistant mutants which were free from thefeedback inhibition of pantothenate kinase byCoA were used as the catalyst (SHIMIZU et al.,1984). In a similar manner, all the intermedi-ates involved in the biosynthesis of CoA fromD-pantothenic acid, i.e., 4b-phosphopanto-thenic acid, 4b-phosphopantothenoylcysteine,4b-phosphopantetheine, and 3b-dephospho-CoA, can be prepared (SHIMIZU and YAMADA,1986, 1989b).

2.4 Pyridoxine (Vitamin B6)

Vitamin B6 compounds, mainly pyridoxineand pyridoxal 5b-phosphate, are exclusivelyproduced by chemical synthesis [ca. 2,500 taP1; major producers, Takeda, Hoffmann-LaRoche, Fuji/Daiichi (Japan)]. They have manypharmaceutical and feed/food applications.Recent chemical and molecular biology stu-dies revealed that 1-deoxy-D-xylulose and 4-hydroxy-L-threonine are the precursors for thebiosynthesis of pyridoxine (TAZOE et al.,2000), but its complete biosynthetic pathway isnot known in detail. Screening for vitamin B6

producers among microorganisms found sev-eral potential strains, such as Klebsiella sp.,Flavobacterium sp., Pichia guilliermondii, Ba-cillus subtilis, Rhizobium meliloti and so on.

Fig. 6. Enzymatic routes for the synthesis of D-pan-tolactone via ketopantolactone. KPL, ketopantolac-tone; KPA, ketopantoic acid. For other abbrevia-tions, see Fig. 4.

vitamins

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