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Revision as of 01:06, 18 September 2015
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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Vitamin B2
Background
Aims
Results
Some microorganisms (e.g. B. subtilis) naturally produce riboflavin in high amount. Make Idli batter microorganisms produce high amount of riboflavin during fermentation to increase the vitamin B2 content of the batter. - Successfully cloned the full riboflavin biosynthesis pathway of B. subtilis in E. coli and made it over-produce vitamin B2 and proceeded to chromosomaly integrate the full pathway in Lactococcus lactis
- BioBrick submitted to the BioBrick Registry: BBa_K1678001, BBa_K1678002, BBa_K1678003, BBa_K1678004
Motivation
Riboflavin derived coenzymes (FMN and FAD) are the cofactors of numerous oxydo-reduction enzymes and are also used in the energy transduction process. It is also used in antioxidative reactions and is required for the metabolism of several other vitamins.
Riboflavin deficiency is a rampant problem in India. For socio-economic reasons, indians are not consuming riboflavin-rich food (dairy products, offal, eggs, almonds...). Only 13% of the households meet the riboflavin dietary requirements and more than 70% of women and children of low-income groups (2009 Indian Council of Medical Research report) and 66% of children from middle-income groups have biochemical evidence of riboflavin deficiency (S. Swaminathan & al., European J. Clin. Nut., 2013).
The most recognizable manifestation of advanced riboflavin deficiency are orolingual, dermal, corneal and hematological. In earlier stages, riboflavin deficiency decreases psychomotor abilities, induces fatigue as well as itching and burning in the eyes.During pregnancy, a riboflavin deficiency can lead to limb-reduction in infants (M. S. Bamji et al., Bulletin of Nutrition Foundation of India, 1993).
Here is described how we adapted the riboflavin biosynthesis pathway from a natural riboflavin overproducer, Bacillus subtilis to engineer Lactic Acid Bacteria (LAB) in order to make them produce high quantities of riboflavin while fermenting Idli batter.
Design
Riboflavin is synthesized by Plants, Bacteria and Fungi. Two fungi, Ashbya gossypii and Candida famata and a Gram positive bacteria, Bacillus subtilis are industrially used as riboflavin overproducers (K.-P Stahmann & al, Appl Micr. Biotech., 2000).
As our team decided to focus on the Idli batter, we reviewed the different publication about the Idli batter microbiome. A broad range of micro-organisms have been characterized in Idli batter, mainly, Gram positive bacteria such as Leuconostoc, Weissella, Pediococcus, Lactobacillus (C. Saravanan & al, J Food Sci Technol, 2015) and also Lactococcus ('Applied Microbiology', Sanjai Saxena).
Even if some of these bacteria are natural producer of riboflavin, their production was not big enough to meet the nutritional requirements.LAB, as like Lactococcus and Lactobacillus, are used worldwide to ferment food. As both are present in Idli batter, we choose to work on Lactobacillus plantarum, which is commonly found in Idli and other fermented food, has been sequenced and for which several engineering protocols have been elaborated.
Our goal was to make it produce as much riboflavin as possible. Thus, we decided to transfer Bacillus subtilis pathway, which is more closely related to Lactobacillus than the two other overproducers, into Lactobacillus plantarum.
Also, to prevent most of the horizontal gene transfer events and to make the insertion more stable and resilient, we decided to integrate the genes coding for the enzymes of the pathway into the chromosome.First, we identified the different enzymes required to produce riboflavin in B. subtilis (K. -P. Stahmann, Appl. Mic.Biotech, 2000)(JB Perkins, J. of Ind. Mic. & Biotech., 1999)(A. G. Vitreschak, Nuc. Acid Res., 2002).
The riboflavin biosynthesis pathway is detailed bellow.
GTP: Guanosine triphosphate
DARPP: 2, 5-diamino-6-ribosylamino-4 (3H)-pyrimidinone-5'-phosphate
ARPP: 5-amino-6-ribosylamino-2,4 (1H, 3H)-pyrimidinone-5'-phosphate
ArPP: 5-amino-6-ribosylamino-2,4 (1H, 3H)-pyrimidinedione-5'-phosphate
ArP: 5-amino-6-ribitylamino-2,4 (1H, 3H)-pyrimidinone
Ribu-5-P: Ribulose 5 Phosphate
DHBP: 3, 4-dihydroxy-2-butanone 4-phosphate
DRL: 6, 7-dimethyl-8-ribityl-lumazine
RibA: GTP cyclohydrolase II / 3,4-dihydroxy-2-butanone 4-phosphate synthase
RibD: Pyrimidine deaminase/reductase
RibE: Riboflavin synthase, beta-chain
RibT: Riboflavin synthase, alpha-chain
Specification
From the lab to the world
Quality control