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Production of L-lysine from starch by Corynebacterium glutamicum displaying -amylase on its cell surface

Importance of Corynebacterium glutamicum. In 1950's , Corynebacterium glutamicum was shown to produce large quantities of glutamic acid (Kinoshita et al., 1957)Gram-positive rods and aerobic Produces amino acids such as L-glutamic acid ,L-lysine, L-threonine and L-isoleucine from aspartateGenome of Corynebacterium glutamicum ATCC 13032 was sequenced (Kalinowski et al., 2003)..

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Production of L-lysine from starch by Corynebacterium glutamicum displaying -amylase on its cell surface

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    1. Production of L-lysine from starch by Corynebacterium glutamicum displaying ?-amylase on its cell surface Toshihiro Tateno ,Hideki Fukuda & Akihiko Kondo Appl. Microbiol. Biotechnol. (2007) 74:1213–1220

    2. Importance of Corynebacterium glutamicum In 1950’s , Corynebacterium glutamicum was shown to produce large quantities of glutamic acid (Kinoshita et al., 1957) Gram-positive rods and aerobic Produces amino acids such as L-glutamic acid ,L-lysine, L-threonine and L-isoleucine from aspartate Genome of Corynebacterium glutamicum ATCC 13032 was sequenced (Kalinowski et al., 2003).

    4. Organic acids such as succinic acid and lactic acid produced under oxygen deprived conditions by adding bicarbonate (Okino et al.,2006) Ethanol production by expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhb) from Zymomonas mobilis ( Inui et al.,2004)

    5. Cell Surface Display Examples of proteins used for heterologous expression: S.aureus protein A, Outer membrane protein (Omp A) ( Samuelson et al., 2002) Production of lactic acid from starch from Lactobacillus casei using PgsA-Amy A fusion protein ( Narita et al., 2006)

    6. Pgs A – Amy A fusion protein Pgs A from Bacillus subtilis , is a member of the poly-?-glutamate synthetase complex ( Kubota et al.,1993) Pgs A is a transmembrane anchor and can be used to fuse proteins on its C-terminus ( Ashiuchi et al., 2001) Amy A from Streptococcus bovis 148 was fused to Pgs A from the N-terminus.

    7. Plasmid Construction sacB selection is on basis of the positive selection system. It was determined that expression of sacB in presence of sucrose was lethal to Corynebacterium glutamicum. Cells which have undergone recombination event would have sacB deleted and would be able to grow on sucrose( Jager et al.,1992). Hence selection for the two recombination events were performed by sacB selection and homoserine auxotrophy. The recombinants were grown on MM solid media with 0.5 g/l L-homoserine and 10g/l sucrose, after kanamycin resistance selection. The recombinants were denoted as ?hom::HPA.sacB selection is on basis of the positive selection system. It was determined that expression of sacB in presence of sucrose was lethal to Corynebacterium glutamicum. Cells which have undergone recombination event would have sacB deleted and would be able to grow on sucrose( Jager et al.,1992). Hence selection for the two recombination events were performed by sacB selection and homoserine auxotrophy. The recombinants were grown on MM solid media with 0.5 g/l L-homoserine and 10g/l sucrose, after kanamycin resistance selection. The recombinants were denoted as ?hom::HPA.

    8. Results A] Western Blot

    9. B] Immunofluorescence microscopy

    10. C] Flow-cytometric analysis

    11. D] Fermentation analysis The medium used is SMMYE-1 contained soluble starch 50 g/l ,L-homoserine 0.5g/l, growth factors and the micronutrients. The medium used is SMMYE-1 contained soluble starch 50 g/l ,L-homoserine 0.5g/l, growth factors and the micronutrients.

    12. ?-Amylase activity and Cell growth Both the non-transformant ( C.glutamicum) and ?hom::HPA were grown on SMMYE-1 which contained starch as the sole carbon source ( 50 g/l) and homoserine 0.5 g/l ( in case of the recombinant).Both the non-transformant ( C.glutamicum) and ?hom::HPA were grown on SMMYE-1 which contained starch as the sole carbon source ( 50 g/l) and homoserine 0.5 g/l ( in case of the recombinant).

    13. ?-Amylase activity assay

    14. Changes in L-lysine and starch concentration L-lysine concentration was determined by reverse phase HPLC and the total sugar concentration was determined by TLC.L-lysine concentration was determined by reverse phase HPLC and the total sugar concentration was determined by TLC.

    15. Discussion Yield and production of L-lysine was found to be 18.89% and 6.04g/l at 30 0C , pH 7.0 The OD 600 value of ?hom::HPA was 4.2 times higher than wild type using soluble starch as the sole carbon source After 30 h of incubation, higher molecular weight sugars were still present, since the conditions were not optimized in terms of Amy A activity and also Amy A cannot hydrolyze ?-1,6-glycosidic bonds.

    16. Conclusion An efficient direct conversion of starch to L-lysine was achieved by displaying Amy A on the cell surface. Thus simultaneous saccharification and fermentation was demonstrated.

    17. Future Prospects The yield of L-lysine can be further increased by co-expression of glucoamylase with Amy A , since ?-amylase cannot hydrolyze ?-1,6-glycosidic bonds in starch.

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