Metabolic pathway engineering for the production of L-Ascorbic acid

- Santosh G. Jadhav

Index
‡ Introduction ‡ Microbial pathway i. Bacterial pathway

ii. Algae pathway ‡ ‡ ‡ Plants pathway Animal pathway Reichstein pathway
2

Introduction
‡Ascorbic acid or L-Ascorbic acid, L-xyloAscorbic acid, L-threo-hex-2-enoic acid Lactone is IUPAC name for Vitamin C (ascorbic acid). ‡In 1928 ascorbic acid is isolated SzentGyorgi and after 5 year chemical structure was determined by Haworth and Hirst.

Figure 1 Chemical structure of Ascorbic acid

Need of study: ‡Scurvy is occurred due to deficiency of Ascorbic acid. ‡Fatal diseases like cancer and cardiovascular. ‡Due to antioxidant property, It used in pharmaceutical, cosmetics, and food industries. ‡Ascorbic acid is taken by human as vitamin.

3

Bacterial pathway
‡ Six metabolic pathway for bacterial fermentation for production of 2-keto-L-gulonic acid: ‡ Sorbitol pathway. ‡ L-idonic pathway. ‡ L-gulonic pathway. ‡ 2-keto-D-gulonic pathway. ‡ 2,5-diketo-D-gulonic acid pathway. ‡ 2-keto-L-gulonic acid pathway.
4

Figure 2 Metabolic pathways for Ascorbic acid from bacteria is given by Bremus et al,(2006)

Commercially most advantages methods are: ‡ Sorbitol pathway. ‡ 2-keto-D-gulonic acid pathway.
5

Sorbitol pathway
‡ Many strains of bacteria are used. These are pseudomonas and acetobacter. ‡ Catalyze the oxidation of D-sorbitol to 2-KLG via series of membrane bound dehydrogenase for L-sorbosone. ‡ L-sorbosone oxidized by membrane bound dehydrogenase or cystolic sorbosone dehydrogenase. ‡ 60g/l with 60% conversion by Glucano oxydans.
6

‡ Presence of cystolic reductase. ‡ Membrane bound dehygenate recombinant with glucanobacter oxydans. e.g. Acetobacter liquifacian ‡ Yield from L-sorbose is 68-81% and from L-sorbosone is 23-83%.

7

2-keto-D-gulonic acid pathway
‡ No bacterial strains are effective. ‡ Three steps towards conversion I. D-glucose to 2-keto-D-gulonic acid by Acetobacter melanogenus and pseudomonas albosesamae. II. Oxidation of 2-keto-D-gulonic acid by using Bacterium hoshigaki and Bacterium gluconicum. III. Oxidation of 2,5-diketo-d-gluconic acid by using Brevibacterium, pseudomonas and Brevibacterium ketosoreductum.

8

‡ Erwina strain followed by Corynebacterium. ‡ Yield is 85% 2-KLG with 10.5g/l of concentration. ‡ Erwina citreus expressed in cytoplasmic 2,5-DKG reductase from corynebacterium yields 19.8g/l with49% conversion.

9

Conversion of 2-KLG to Ascorbic acid
1.

Multistep process. Esterification of 2-KLG under strong acidic condition to methyl-2-keto-l-gulonate (MeKLG). MeKLG with base to produce metal ascorbate salt Metal salt treated with acid to form ascorbic acid cyclization of 2-KLG.

‡

‡ ‡

2. Single step process comprise acid-catalyzed

10

Pathway of Algae
‡ Heterotrophic green microalga chlorella pyrenoidosa yield 40mg/l but after mutation and optimizing yield 2 g/l ascorbic acid. ‡ Colorless microalga Prototheca moriformis. ‡ Prototheca used for mutation of many strains. ‡ The biosynthesis pathway is similar like in plants.
11

Figure 3 metabolic pathway for Ascorbic acid by algae is given by Bremus et al,(2006).

12

Pathway for plants

Figure 4 Metabolic pathway for Ascorbic acid by plants is given by Hancock and Viola (2002).

13

‡ Phosphomannose isomerase (PMI) catalyze fructose to dmannose. ‡ PMI from E. coli used. ‡ Conversion of d-mannose-6-phosphate to d-mannose-1phosphate by Phosphomannose mutase (PMM). ‡ GDP-D-mannose from d-mannose-1-phosphate by GDPD-mannose pyrophosphorylase (GMP). ‡ GDP-D-mannose convert to GDP-L-galactose reversible double epimerization using GDP-D-mannose -3,5epimerase (GME).
14

‡ GDP-L-galactose converted to L-Ascorbic acid. ‡ GDP-L-galactose broken down to L-galactose -1phosphate hydrolyze to L-galactose by GDP-Lphosphorylase. ‡ L-galactose then oxidize in two steps: I. Cystolic NAD-dependant L-galactose dehydrogenase at C1 form L-galactono-1,4-lactone. II. L-galactose-l-dehydrogenase at C1/C3 to ascorbic acid.
15

Pathway for animals

Figure 5 metabolic pathway for production of Ascorbic acid given by Linster and Schaftingen (2006).

16

‡ Formation of glucuronate from UDP-glucuronate:  Cleavage of UDP-glucuronate to glucuronate-1phosphate followed by dephosphorylation of latter by glucuronate-1-phosphatase.  Formation of glucuronidated intermediate followed by hydrolysis by -glucuronidase or esterase.  Direct hydrolysis of UDP-glucuronate to UDP and glucuronate
17

‡ Glucuronate reductase:  Reduction of D-glucuronate to L-gulonate by NADPH- dependent reductase.  Aldo-keto reductase.  D-glucuronate and D-glucurolactone convert to Lgulonate and L-gulonolactone.

18

‡ Urono and gulonolactonase: 
Conversion D-glucuronate to L-gulonolactone requires

reductase and lactonase. 
Three different type of lactonases act on sugar  6-phosphoglucanolactonase:  Uronolactonase:  Aldolactonase: metal dependant, best with Mn+2, present

in cystol and hydrolyzes number of lactone. Therefore take part in Ascorbic acid synthesis.
19

‡ L-gulonolactone oxidase:  Catalyze the L-gulonolactone to L-ascorbate with H2O2 production aerobically.  The intermediate formed 2-keto-l-gulonolactone isomerizes to L-ascorbic acid.

20

Reichstein process

Figure 6 metabolic pathway for production of Ascorbic acid by animal is given by Hancock and Viola (2002).

21

‡ D-sorbitol: i. Catalytic hydrogenation D-glucose at 140-1500C and 80-125 atm. ii. Minimal formation of D-mannitol and L-idiotol. ‡ L-sorbose: i. Obtained in pyranose form. ii. Gluconobacter oxydans strain used. iii. Large scale operation carried out at 4-6 PH and 30-350C. ‡ 2,3:4,6-Di-O-isopropylidene- -l-sorbofuranose: i. Acetone and sulphuric acid as a catalyst and dehydrating agent at low temperature (40C). ii. 2,3-O-isopropylidene- -l-sorbofuranose and 1,2-Oisopropylidene- - l-sorbopyranose are side products.
22

‡ i.

ii.

iii. iv. v.

2, 3:4, 6-Di-O-isopropylidene-2-ketol-gulonic acid: Oxidation of 2,3:4,6-Di-O-isopropylidene- -l-sorbofuranose to 2,3 : 4,6-di-O-isopropylidene- 2-keto-l-gulonic acid in dilute NaOH with KMnO4. Less expensive methods fro oxidation are: sodium hypochlorite, electrochemical oxidation, catalytic air oxidation. With hypochlorite and nickel chloride or sulphate >93% yield obtained. Electrochemical oxidation nickel or nickel peroxide electrode in alkaline solution (sodium hypochlorite). With air or oxygen and metal (palladium or platinum) in alkaline solution good yield is achieved.

23

‡ i.

L-Ascorbic acid: 2,3:4,6-di-O-isopropylidene-2-keto-l-gulonic acid to lascorbic acid achieved in two way.

a) Conversion to 2-KLG followed by esterification with methanol and base catalyses cyclization of methyl ester. b) Acid-catalyzed cyclization to ascorbic acid directly from 2keto-l-gulonic acid. ‡ Downstream processes: removing impurities, decoloration, and crystallization.

24

Comparison of different pathway
Serial number 1 2 3 4 5 Pathway Bacterial Algae Plants Animal Reichstein pathway D-sorbitol 2-KDG Conversion (%) 60 49 90 Yield (%) 68-81 85 60 Concentration (g/l) 60 19.8 2 -

‡ i. ii. ‡ ‡

Reichstein process is used, High conversion. Inexpensive raw material used for production. Drawback: Require high temperature and pressure in intermediate steps. Modification: The intermediate are biosynthetically produced using genetically modified strain (Gluconobacter Oxydans).
25

Conclusion
Reichstein process used for production of Ascorbic acid from D-glucose due to high conversion and inexpensive raw material.

26

References
1. Bernd Oster and Ulrich Fechtel, E. Merck, Darmstadt, Federal Republic of Germany, (2003) ULLMANN¶S Encyclopedia of Industrial Chemistry (Wiley- VCH), sixth edn. Vol. 38:218-231. 2. Carole L. Linster and Emile Van Schaftingen (2006) Biosynthesis, recycling and degradation in mammals, FEBS Journal, 274:1±22. 3. Christoph Bremus, Ute Herrmann, Stephanie Bringer-Meyer, Hermann Sahm (2006) The use of microorganisms in l-ascorbic acid production, Journal of Biotechnology, 124:196±205. 4. Glen L. Wheeler, Mark A. Jones and Nicholas Smirnoff (1998) The Biosynthetic Pathway of Vitamin C in Higher Plants, Nature, vol. 393:365-369. 5. Robert D. Hancock and Roberto Viola (2002) Biotechnological approaches for L-ascorbic acid production, Trends In Biotechnology Vol.20 No.7:299-305. 6. Shrikant A. Survase, Ishwar B. Bajaj and Rekha S. Singhal (2006) Biotechnological Production of Vitamins, Food Technology Biotechnology, 44 (3): 381±396. 7. Takahiro Ishikawa, John Dowdle and Nicholas Smirnoff (2006) Progress in manipulating ascorbic acid biosynthesis and accumulation in plants, Physiologia Plantarum, 126: 343±355.

27

Thank you !!!

Sign up to vote on this title
UsefulNot useful