Overview

Our iGEM Bordeaux team 2013 has worked on the development of new lactic ferments able to produce flavours on their own and resveratrol also, a molecule which is considered to be responsible of good effects of red wine. This project will allow on the long run an easier production of flavoured yogurts with beneficial effects on the organisms and medical properties without using chemical substances. On top of that yogurt can be produced in an industrial way or on a small scale , the project can be used on one of those two scales.

The yoghurt's choice

The economic issues have ever been an important concern for agronomic industries. How to produce better, faster and cheaper? New technologies help us to improve yields and decrease production rates. Progress in Health and Biotechnologies fields let us to intend fortified food with healthy and curative compounds.

« Yogurt or yoghurt or yoghourt is a fermented milk product (soy milk, nut milks such as almond milk, and coconut milk can also be used) produced by bacterial fermentation of milk. The bacteria used to make yogurt are known as "yogurt cultures". Fermentation of lactose by these bacteria produces lactic acid, which acts on milk protein to give yogurt its texture and its characteristic tang » Wikipedia.

From this point of view, yoghurt appears as an ideal support to begin an Igem project. In spite of adding synthetic flavours, we could modify bacteria to make them produce flavours directly in yoghurt. And what for dyes ? Already in yoghurt in too ! And why not adding drugs ? There are infinite possibilites. You will just need milk and modified bacteria to make a « SUPER YOGHURT ».

Our iGEM Bordeaux team 2013 has worked on the development of new lactic ferments able to produce flavours on their own and resveratrol also, a molecule which is considered to be responsible of good effects of red wine. This project will allow on the long run an easier production of flavoured yogurts with beneficial effects on the organisms and medical properties without using chemical substances. On top of that yogurt can be produced in an industrial way or on a small scale, the project can be used on one of those two scales.

Lactobacillus bulgaricus and Streptococcus thermophilus

1. Microbiology of fermentation

Lactic fermentation is the main step that can ensure the transformation of liquid milk into a yoghurt. The two lactic bacteria used, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus work together in symbiosis.

At the beginning of the fermentation S.thermophilus develop first. Converting lactose into lactic acid, these bacteria provoke a decrease of the characteristic pH of yoghurts. They also synthesize compounds like formic acid or carbon dioxide which increase the growth of L. bulgaricus. Lactobacillus hydrolyze the casein from the milk thanks to a protease bound to the cell wall. Products of this hydrolysis, after several reactions, form amino-acids and in particular Valine which is not present in sufficient quantity in the milk at the beginning of the fermentation. S. thermophilus has a lower protease activity and get benefit from the presence of L.bulgaricus. Thanks to this joint action, the two species grow rapidly and metabolize enough lactose into lactate so that the fermentation ends in 3 to 4 hours instead of 12 to 16 hours if they were alone.

2. Transformation of L. bulgaricus by electroporation

3. Quorum Sensing and regulation

When ingesting yoghurt, the two ferments react in a different way. S.thermophilus is destroyed by the digestive system whereas L. bulgaricus resists different pressure imposed by this digestion (variation in pH, enzymes). L. bulgaricus stays some time into the intestine before being evacuated. We wanted to use this fact to create a quorum sensing system allowing to stop useless synthesis of colors or flavour once the yoghurt has been eaten.

Different synthetic pathway of colors and flavors 

1. Dyes (red pink orange yellow)

Red pigment : lycopene

Lycopene is a red compound of the terpene family from Farnesyl phyrophosphate (a substrate present into L.bulgaricus).

Terpen pathway : to get red

Three enzymes are required to get lycopene : crtE, crtB and crtI. Those enzymes already exist in biobricks (Edinburgh 2007-2008): BBa_K118002 and BBa_K118003. Our goal was to reuse these biobricks, make them express in E.coli, and then transform L.bulgaricus with a constitutive promotor of lactic bacteria. To construct the biobricks, the coding sequence dxs of E.coli JM109 have been added to increase yields of carotenoids.
The biobrick from Edinburgh 2008 have been reused: BBa_K118004 : rbs + dxs

Four new biobricks have been designed :

• BBa_K1148000 : crtE under hlbA promoter (phlbA)
• BBa_K1148001 : crtB under hlbA promoter (phlbA)
• BBa_K1148002 : crtI under hlbA promoter (phlbA)
• BBa_K1148003 : crtE + crtB + crtI under hlbA promoter (phlbA)

Orange and yellow pigments

Orange and yellow colors can be acquired from lycopene with two more enzymes.

Terpen pathway : to get orange and yellow

Two new biobricks have then been designed:

• BBa_K1148004 : crtY under hlbA promoter (phlbA)
• BBa_K1148005 : crtZ under hlbA promoter (phlbA)

2. Purple, Dark green and Light green

Three new biobricks have been designed, based on existing biobricks (BBa_K274002 (purple), BBa_K274003 (dark and light green)).

• BBa_K1148006 : VioA-E under hlbA promoter (phlbA)
• BBa_K1148007 : VioABDE under hlbA promoter (phlbA)

Betanine

Our iGem team had the idea to use colour and dyes from nature to create a new yogourt with all the benefits of natural compounds.
Here are two examples :
Betanine, one of the major compounds of the beet, is commonly used as a food additive (E162), is a natural antioxidant and is well assimilated by the organism. Moreover, the colour of this pigment varies with the pH, an important fact to get different colours. The idea of designing a kit, with modified lactobacillus which produce betanine is interesting, for a use at home.

From the knowledge of the pathway of Lactobacillus delbrukei subsp. Bulgaricus, we have seen that leucodopachrome is produced by the bacteria. Moreover two enzymes: cyclo-DOPA 5-O-glucosyltransferase and betanidin 6-O-glucosyltransferase can produce betanine from leucodopachrome. The strategy of the team was to insert a plasmid with one of these two enzymes in Lactobacillus, to make it produce betanine.

Plasmid with the cyclo-DOPA 5-0-glucosyltransferase gene: BBa_K114820: CDOPA5GT under hLbA promoter

Plasmid with the betanidin 6-0-glucosyltransferase gene: BBa_K114821: 6-GT G9797 under hlbA promoter

Raspberry ketone

Raspberry ketone is the second compound we would like to make bacteria produce. It is the most expensive natural dye in the food industry. The advantage here is to have a cheaper dye, less difficult to extract and produce directly in the yoghourt.

(Source: iGEM project of Munich 2012)

From the first product who is the 4-coumaroyl-CoA, two enzymes are necessary to obtain the Raspberry ketone. The first one is the p-hydroxyphenylbut-3-ene-2-one synthase from Rubus idaeus or Rheum palmatum. The second one is p-hydroxyphenylbut-3-ene-2-one reductase.
After getting the genes coding for these enzymes, two plasmids are created and inserted in Lactobacillus in order to get this new pathway.

Plasmid with the p-hydroxyphenylbut-3-ene-2-one synthase gene: BBa_K114822: CHEBI: 68636 under hLbA promoter

Plasmid with the p-hydroxyphenylbut-3-ene-2-one reductase gene: BBa_K114823: gene coding for p-hydroxyphenylbut-3-ene-2-one reductase under hlbA promoter

3. Flavors (red fruits...)

Mono-terpene pathway

This pathway can be used to produce several compounds adding natural flavor into the yoghurt.

Limonene

This citrus fruit flavor is obtained thanks to lymonene-1-synthase (4.2.3.16). The substrate is Geranyl-PP, also used in the terpene pathway (for red, orange colors). The biobrick has been designed (BBa_K801061)
BBa_K1148008 : the gene for limonene synthase 1 under hlbA promoter (phlbA)

Terpineol

The pine flavor is obtained thanks to 1,8-cineole synthase (4.2.3.108). The substrate is Geranyl-PP. This enzyme can be found in Arabidopsis Thaliana.
BBa_K1148009 : TPC-CIN under hlbA promoter (phlbA)

Myrcene

The lavender flavor is obtained thanks to myrcene synthase (4.2.3.15). The substrate is Geranyl-PP. This enzyme can be found in Arabidopsis Thaliana.
BBa_K1148010 : TPS10 under hlbA promoter (phlbA)

Geraniol

The rose flavor is obtained thanks to monoterpenyl-diphosphatase (3.1.7.3). The substrate is Geranyl-PP.
BBa_K1148011 : the gene for monoterpenyl-diphosphatase under hlbA promoter (phlbA)

The Resveratrol

To go further in the creation of a “natural homemade colored yoghurt” kit, why not imagine yoghurt with benefits for health?
Resveratrol quickly appears as an ideal molecule for a nutriceutical.

However, bacteria cannot naturally produce resveratrol. That is why in this part of our project, we wanted to introduce resveratrol pathway in Lactobacillus.
In this purpose, we focused on the enzymes of this pathway.
There are two indispensable enzymes to make bacteria produce resveratrol.

Biosynthetic pathway of resveratrol. The biosynthesis of resveratrol by the coupling of p-coumaric acid to CoA by the 4CL enzyme. Subsequently, coumaroyl-Co1 is converted into resveratrol by sequential addition of three malonyl-CoA units with the release of carbon dioxide.
Production of Resveratrol in Recombinant Microorganisms; Jules Beekwilder*, Rianne Wolswinkel, Harry Jonker, Robert Hall, C. H. Ric de Vos and Arnaud Bovyhttp://aem.asm.org/content/72/8/5670.full

4-coumarate, coenzyme A ligase (4 CL) and Stilbene Synthase (STS) enzymes have to be expressed in bacteria.

We based on Rice Igem Project of 2008 to start our project. We needed to know if 4CL and SLC enzymes were already available to make the genetic construction.
Part: BBa_K122012 of Rice University consists on the fusion of 4CL and STS enzymes.
First of all, we needed to get the enzymes biobricks before express them in bacteria.
In this purpose, we wanted to design new biobricks. Grape is well known to produce an important rate of resveratrol. That is why wanted to extract these enzymes from grapes.

To get these enzymes from grape, we obtained cDNA from a reverse transcription on grapes pericarps extracts. We performed a PCR on the two genes: 4CL and STS in order to assemble them within a single biobrick under the control of a promoter (R0011 http://parts.igem.org/Part:BBa_R0011 ) induced by the presence of lactose in the growth medium.

Scale-up to artisanal and industrial scale

Industrial scale

Freeze-drying and lactic ferments kits