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RANJAN KOIRALA PHD Researcher BIOTECHNOLOGY NAST

Introduction
Heterologous: Synthesizing Foreign metabolites Native: Regular metabolites Mannitol is reduced form of Fructose and is produced by

variety of microorganisms (Bacteria, yeast & Molds) Organism maintain redox balance by the production of mannitol. Physiological function as osmolyte and can serve as protective agent. Mannitol enhances survival of Lactobacillus lactis cells during drying process. (LAB used as starter culture may thus enhanced by MANNITOL) Other various function in the laboratory.

LAB
LAB (Lactic Acid Bacteria) is extensively used in Dairy

Industry. Use of mannitol producing Lactococcus lactis may result in fermented products with extra value, since mannitol is assumed as several beneficial effects as food additive. Serve as antioxidant. Low calorie sweetners that replace sucrose. Other various microbial biotechnological benefits.

Glucose fermentation by LAB for mannitol biosynthesis

Homofermentative LAB (Metabolic Engineering Strategy)

Heterofermentative LAB

Production of M1Pase Production of M1PDH Eg. L. lactis

(Limited production due to active mannitol transport)

Single conversion by Mannitol dehydrogenase eg. Leuconostock mesenteroides

Metabolic Engineering of L. lactis for over production of mannitol


M1PDH from M1Pase cloned Lactobacillus Both were downstream plantarum compared to M1PDH with the But alone did from Eimeria control strain. not produce tenella mannitol

M1PDH

Lactobacillu s plantarum

Eimeria tenella

M1Pase

Building the Desired Genes


ON/OFF Switch PROMOTER INTRON Makes Protein CODING SEQUENCE Gene of Interest stop sign
poly A signal

Selectable Marker Gene

Plasmid DNA Construct


bacterial genes
antibiotic marker replication origin

Materials & Methods


Lactococcus lactis strains, Plasmids, Media

Strains grown at 300C in M17 broth (oxoid) + glucose. When semi-anaerobic : cells grown in batch culture in 50 ml tubes

without aeration. When aerobically: cells grown shaking flasks with baffles were used. When applicable Chloramphenicol & Erythromycin at 10& 5 l/ml supplemented. OD taken for growth measurement at 600 nm with an ultraspec 2000 spectrophotometer. For induction of M1PDH & M1Pase activity: 0.1-10 ng of nisin/ml added to a growing culture with OD600 about 0.5 or more. Comparative study of mannitol production by analyzing respective enzyme activity after genetic engineering of respective genes.

Construction of Plasmid pWW002 &pWW003


Donor Organis m Control Strain Eimeria. Tenella Gene of Amplificat Interest ion mode of Gene mtlD and M1Pase M1Pase Vector Resultin g Plasmid pNZ8148 Cloned to strain Remarks

NZ9000 Control strain

PCR from E. pNZ8148 pWW002 NZ9000 Nisin inducible coli expression expression plasmid vector PQE60 Not mentioned (previous work) pNZ8148 pWW003 NZ9000 M1Pase gene cloned downstream to mtlD for simultaneous expression of mannitol

Lactobacil lus plantarum

M1Pase & M1PDH

Analysis of fermentation product & glucose consumption


Growth culture of L. lactis taken
Centrifuged at 10000 g Supernatant stored at -200C until further analysis

lactate, acetate, formate, glucose, mannitol, ethanol,

2,3-butandiol and acetoin etc. were analyzed by quality and quantity.

Preparation of Cell Extracts


50 ml of cell culture (OD600 aprox. 1.2or 2 hrs

incubation after nisin induction) Centrifuged at 40C, 20 min at 2000 g Crude enzyme extracts prepared with MES (morpholine ethane sulphonic acid) buffer (pH 7) Protein content detected by BCA protein assay with BSA (Bovine serum albumin standard)

Enzyme Assay
Crude enzyme extracts taken to analyze two different

enzymes. M1PDH analyzed using sodium phosphate buffer method and EA determined in mol-1 mg of protein-1 M1Pase Enzyme activity determined by Sigma diagnostic Inorganic Phosphate Kit. M1PDH analyzed using sodium phosphate buffer method and EA determined in mol-1 mg of protein-1

Result and Discussion


Overexpression of the M1Pase gene and mtlD.
Nisin-dependent mannitol production by

overproduction of M1PDH and M1Pase. Fermentation patterns. M1Pase-dependent mannitol production. Improved mannitol production by L. lactis NZ9000(pWW003).

Mannitol production related to M1Pase and M1PDH activity in L. lactis


L. Lactis strain Nisin (ng/ml) Enzyme Activity ( mol min-1 mg of protein-1)
M1Pase NZ9000(pNZ8148) 0 1 0 1 0 1 <0.01 <0.01 0.02 0.90 <0.01 0.27 M1PDH <0.01 <0.01 <0.01 <0.01 <0.01 2.2 <0.1 <0.1 <0.1 <0.1 <0.1 2.5

Mannitol (mM)

NZ9000(pWW002)

NZ9000(pWW003)

the M1Pase gene of E. tenella and the M1PDH gene mtlD from L. plantarum. Cells were grown anaerobically on M17 broth supplemented with 0.5% glucose; M1PDH and M1Pase production was induced with 0, 0.1, 0.3, 1.0, 3.0, and 10 ng of nisin/ml at an OD600 of 0.5; and cells were harvested 2 h after induction. Mannitol production during growth on glucose was recorded. L. lactis strains used are as follows: NZ9000(pWW003) (A and D), HWA217(pWW003) (B and E), and NZ9010(pWW003) (C and F). Black bar, M1Pase; grey bar, M1PDH (A to C); , 0; , 0.1; ,

0.3;

, 1.0;

, 3.0; and

, 10 ng of nisin/ml.

Product formation by L. lactis strains overproducing E. tenella M1Pase and L. plantarum M1PDHa
1507.pdf

Conclusion
The combined overproduction of M1PDH and M1Pase has

proven to be a successful strategy for obtaining a mannitol producing L. lactis. This work presents the first example of high and stable mannitol production in growing L. lactis cells, in contrast to the mannitol production observed for resting L. lactis cells. The results shown here emphasize the importance of M1Pase activity for mannitol production by L. lactis and indicate that a L. lactis strain deficient in LDH activity and with high M1PDH and M1Pase activity would be a good candidate for in situ mannitol production in food products.

Reference
APPLIED AND ENVIRONMENTAL MICROBIOLOGY,

Mar. 2005, p. 15071514 Vol. 71, No. 3 0099-2240/05/$08.000 doi:10.1128/AEM.71.3.1507 1514.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved.

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