PRODUCTION OF VITAMIN C

Group members: Khairul Izwan

Muhammad Arshad

Nik Abd Hafiidz

Nor Hafiza Izzati

Nur Afiqah

COMPANY PROFILE

VITAMIN C

-Water soluble micronutrient

-Important regulator of iron uptake

- Vitamin C is a strong reducing

agent (Aysun, 2009).

SELECTION OF MICROBE

Gluconobacter oxydans

Gram negative bacterium

Simple, non-pathogenic

Large oxidative potential

WHY G. OXYDANS?

O Its colonies’ strains are pale, able to grow in chemically defined medium (Arun et.al, 2001).

O Can be used industrially to produce L-sorbose from D-sorbitol.

O Can be manipulated for the expression of foreign genes (Zhou et.al, 2013).

Enzymatic of enzyme

G.oxydans only produce (SLDH)

but G624 can produce three

enxymes (SLDH,SDH and

SNDH)

Complex reaction.

Oxidation reaction from D-

glucose to 2,5-DKGA-forming

enzyme and the reduction of 2,5-

DKGA reductase are not fully

coorperative

By cloning both genes for SDH and

SNDH from G. oxydans T-100 into

G.oxydans G624, the production of 2-

KLGA could be produced directly

LIMITATIONS

STEPS RECOMBINANT IN GENERAL

Isolate Bacteria Purification of SDH and SNDH

Cloning of genes for SDH and

SNDH

Expression of genes for SDH and SNDH in E.

coli

Preparation of shuttle vectors

Expression pSDH155

i. Isolate bateria

O G. oxydans T-100, a 2-KLGA-producing

strain, and G. oxydans G624 an L-sorbose-

accumulating strain, were isolated from a

Japanese persimmon (kaki) and from a

Japanese peach, respectively.

ii. Purification of SDH and SNDH

O The molecular mass of the purified SDH was approximately 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis. The amino-terminal sequences of the SDH protein (TSGFDYIVVGGGSA)

O The molecular mass of the purified SNDH was approximately 50 kDa as determined by SDS-PAGE analysis. The amino-terminal sequence was determined to be (NVVSKTVXL)

O Interestingly, the gene for the SNDH consisting of 498 amino acids was found to be located in the upstream portion of the SDH gene whose translational initiation codon ATG overlapped with the termination TAA of the SNDH (Fig.1) (saito et. al.).

iii. Cloning of genes for SDH and SNDH

O The genomic DNA isolated from G.oxydans T-100 was

partially digested with MboI. The 8- to 22-kb DNAs

were separated by sucrose gradient

ultracentrifugation and cloned into the BamHI site of

λ phage vector

O A λ phage DNA positive to the probe was digested

with SalI and EcoRI and analyzed by Southern

blotting. An approximatelyb 6-kb DNA fragment that

hybridized to the probe was isolated and cloned into

pUC19 between the SalI and EcoRI sites to give

pUC19SD5.

iv. Expression of genes for SDH and SNDH in E. coli

O The recombinant E. coli JM109 transformed with

pUC19SD5 was cultivated

O The E. coli JM109/ pUC19SD5 expressed SDH and

SNDH activity

O It is obvious that the region contains the promoter for

the SNDH and SDH genes. These results indicate that

pUC19SD5 contains the whole DNA for the SDH and

SNDH synthesis system, in accordance with the

expectation from the sequence analysis.

v. Preparation of shuttle vectors pf4

O A plasmid, pF4, was extracted from G. oxydans ,T-100

by the alkaline-SDS method and purified by

ultracentrifugation with CsCl.

O It is ligated with pHSG298 (Takara Shuzo) at the HindIII

site to prepare a shuttle vector, designated pFG15A

O pFG15A was introduced to G. oxydans G624, which

accumulates L-sorbose in the presence of D-sorbitol.

Preparation of shuttle vector pSDH155

O pUC19SD5 was digested with SalI and BglII and

filled in with DNA polymerase. The resulting 7-kb

fragment was self-ligated and then digested with

EcoRI and PstI to give a 4.6-kb DNA carrying the

promoter, genes for SNDH and SDH, and the

terminator.

O pFG15A was digested with EcoRI and PstI, and the

resulting 7.3-kb DNA fragment was ligated to the

4.6-kb DNA fragment to give pSDH155

vi. Expression pSDH155O G. oxydans G624 was transformed with pSDH155 and

cultivated in the presence of D-sorbitol.

O G. oxydans G624 itself showed no ability to produce 2-KLGA.

O The recombinant Gluconobacter, G. oxydansG624/pSDH155, was 2.3-fold more active in the production of 2-KLGA than was G. oxydans T-100

O No unreacted D-sorbitol was detected in the broth. These data show that D-sorbitol was completely converted to L-sorbose and that active SDH and SNDH were expressed by pSDH155 in the recombinant Gluconobacter. Although considerable amounts of L-sorbose remained unconverted and L-idonate was also a by-product , the stoichiometry of the reaction revealed that no catabolites were produced in the fermentation pathway.

MNNG mutation of host strainO To obtain a mutant whose enzymatic pathway from 2-KLGA to

L-idonate was blocked, G. oxydans G624/pSDH155 was treated with MNNG according to the conventional method.

O From one of the mutated, strains, a mutant (IA1069) whose productivity of L-idonate was greatly reduced was isolated (Table 2).

O After treating the mutant with novobiocin to remove the plasmid, a mutant strain, designated NB6939, was obtained. The resultant G. oxydans NB6939 showed no effect on the metabolism of L-sorbose and L-sorbosone (data not shown). It was retransformed with pSDH155. The productivity of 2-KLGA by G. oxydans NB6939/pSDH155 increased by 90% over that by G. oxydans G624/pSDH155, indicating that MNNG mutated the genomic DNA of G. oxydans G624 participating in L-idonate synthesis.

Replacement of the promoter sequence in the shuttle vector

O The effect of the promoter on the productivity of 2-

KLGA, several types of E. coli promoter DNA

containing 235 and 210 regions were synthesized

based on the reported sequences and were inserted

in place of the original promoter DNA (Fig. 4).

O In particular, the production of 2-KLGA by G. oxydans

NB6939/pSDH-tufB1 was 88 mg/ml with 72 h of

fermentation in a broth containing 10% D-sorbitol.

O Preparation of shuttle vector pSDH-tufB1. An NcoIsite in the downstream region of the SDH gene of pSDH155 was destroyed and a new NcoI site was introduced in the upstream region of the SNDH gene by PCR mutation to give pSDH155-NN (11.9 kb). To remove the lacpromoter, originating from pHSG298, pSDH155-NN was partially digested with PvuII. The resulting DNA fragment (11.9 kb) was ligated with an EcoRI linker (Pharmacia), digested with EcoRI, and self-ligated to give pSDH165-NN. The EcoRI-NcoIregion of pSDH165-NN was replaced with synthetic oligodeoxyribonucleotides (TUFB1, -2, -3, and -4) for the E. coli tufB promoter (1) to obtain the desired pSDH-tufB1. ter, terminator sequence.

Suitable environment for microbial growth

O The growing step can be performed in an aqueous

medium which is supplemented with appropriate

nutrients for growth under aerobic conditions.

O The cultivation may be conducted at a pH of about

5.0 to about 8.0.

O A suitable temperature range for carrying out the

cultivation from about 18° C. to about 33° C.

Biochemical reaction involved

Optimum conditions of the culture growth in bioreactor

O culture temperature for growth is preferably at 20-35° C.

O When a microorganism belonging to the genus Gluconobactor is used as a host and SDH and/or SNDH are/is expressed, the pH is preferably 7-7.5

O cells of G. oxydans were grown in medium containing 100 g/l D-sorbitol, 0.5 g/l glycerol, 15 g/l yeast, 2.5 g/l MgSO4.7H2O and 15 g/l CaCO3 in a 2-l baffled shake flask at 30° C. with shaking at 180 rpm.

Medium use in recombinant and operating condition

Purification of SDH and SNDH

O G. oxydans T-100 was cultivated in 20 liters of a broth containing 5% L-sorbitol, 0.5% glycerol, 0.5% yeast extract, and 1.0% CaCO3 at 30 C for 42 h.

Assay of SDH and SNDH activities expressed in E. coli

O E. coli strains were cultivated in 200 ml of a broth (pH 6.8) consisting of 1% Bacto Tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1% glycerol, 0.3% KH2PO4, 0.8% Na2HPO4 z 12H2O, and 1% L-sorbose with (JM109/pUC19SD5) or without (JM109) ampicillin (100 mg/ml) at 258C for 72 h.

Transformation and chemical mutation of Gluconobacter

O The cells of G. oxydans G624 cultivated in 100 ml of MB broth (2.5% D-mannitol, 0.3% polypeptone, 0.5% yeast extract [pH 6.0]) at 258C for 20 h were washed with 10% glycerol and suspended in 1 ml of 10% glycerol to a concentration of approximately 1010 cells/ml. Mutation of G. oxydansG624/pSDH155 by (MNNG) was performed essentially. Each mutant was cultivated in 1 ml of a broth consisting of 5% D-sorbitol, 0.5% yeast extract, and 2% CaCO3 at 308C for 24 h.

ETHICAL ISSUE

O Mainly related to the impact they have on the Earth's biosphere.

O The danger posed by these genetically modified organisms is therefore related both to their dispersal into the environment and to their potential for adaptation to a new environment.

O People are concerned with the release of genetically engineered microorganisms into the environment. The fear is that such organisms may continue to proliferate and it may not be possible to “stop them.”

O The use of genetically engineered organisms in the food we eat. People are worried that genetically engineered organisms may pose an unknown health risk and they feel that it is morally wrong to tamper with the genetics of organisms. This opinion may also apply to genetic techniques such as cloning, stem cell research, and gene therapy.