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.

Genetic sequences of Bacillus subtilis four enzymes were codon optimized for Lactobacillus plantarum on IDT website tool.
The enzyme was expressed by a promoter rather than a single operon expression to allow a precise tuning of the gene transcription. Two metabolic bottlenecks were identified in the pathway (M. Birkenmeier, Biotech. Lett., 2014). The first bottleneck correspond to the GTP cyclohydrolase activity of RibA. Overcoming the first bottleneck creates a second bottleneck corresponding to RibT's lumazine synthase activity.
To promote a differential expression of the four genes, we used the two synthetic Lactobacillus promoters p25 and p48 (respectively medium and strong expressing promoters) (I. Rud, Microbiology, 2006).
RibA expression was therefor promoted by p48, RibD and RibE by p25 and RibT by either p25 or p48.
For translation initiation, we used the consensus RBS for Lactobacillus (Tauer et al. Microbial Cell Factories 2014, 13:150).
T_ldh terminator from L. buchneri lactate dehydrogenase gene was used to stop the transcription (Spath et al. Microbial Cell Factories 2012, 11:141).

Results

The chromosome integration plasmid pKV6 was first altered to allow the insertion of the four genes by Golden Gate Assembly. This new plasmid was called p15.01.
Then, the four genes were successfully inserted in p15.01 and cloned in E. coli.
After cloning, cells began to release riboflavin in their surrounding media, suggesting that the expression system was functionning in E. coli as well. We characterized the functioning of promoter p48 in E. coli and BioBrick it(BBa_K1678004).

Discussion

Though successfully we managed to produce Riboflavin (Vitamin B2) in E.coli, which was the vital obstacle to overcome but due to time constraints this project could have proceeded to introduce this B2 producing plasmid into Lactococcus lactis as mentioned by Christian Solem et al, 2013 and integrate the whole pathway into L. lactis chromosome. We used the same plasmid used by them for repetitive, marker-free, site specific chromosomal integration of foreign DNA, in this case vitamin B2. So for the further development of the project we can proceed to chromosomal integration of the vitamin B2 in Lactococcus and as once the plasmid is integrated, the site can be used for repetitive chromosomal integration. We can integrate Vitamin A and Vitamin B12 pathways in the same way thus probably creating a single cell with multiple vitamins producing capability.