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Team:Edinburgh/Project
From 2008.igem.org
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"Cellulose is the most abundant form of fixed carbon, with 100,000,000,000 tons produced in cell walls by plants each year" [http://www.blackwell-synergy.com/doi/full/10.1196/annals.1419.026 (Wilson, 2008)].
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Engineering bacteria to convert cellulose into starch and to produce the vitamin A precursor, beta-carotene
Currently, much agricultural produce is wasted. Wouldn't it be great if the indigestible parts of crop plants could be made edible, or at least if they could be converted into a biofuel source? - This is our plan!
We will endow bacteria with three novel abilities:
- to degrade cellulose into glucose
- to synthesise starch from glucose
- to synthesise beta-carotene from glucose
1. Cellulose degradation
We plan to incorporate the Cellulomonas fimi endoglucanase genes cenA, cenB and cenC, exoglucanase gene cex and beta-glucosidase into bacteria. Together these genes will be capable of breaking down cellulose into glucose. The proteins will need to be secreted into the medium to act (cellulose being too large for uptake into the cells), and we have 3 plans to enable this:
- Add the Hly secretory pathway into a lab strain of E. coli (K12, JM109). This will involve incorporating hlyB, hlyD and tolC and adding the 3' end of hlyA to our cellulases.
- Create a glucose-sensitive feedback mechanism of cell lysis. Depletion of glucose would cause transcription of our cellulase operon and the transcription factor comK. ComK would bind to a promoter upstream of the phiX174 gene E. E is a short peptide which causes cell lysis.
- Engineer our genes into Bacillus subtilis, in which secretion is much better understand than in E. coli.
2. Starch synthesis
The first phase in synthesising starch make use of the chasis' native glycogen synthesis pathway. The gene glgC (ADP-glucose pyrophosphorylase, catalysing the convertion glucose 1-phosphate and ATP to ADP-glucose and PPi) is responsible for the most rate-limiting step of glycogen synthesis in E. coli. This is because the protein is negatively regulated by PPi. Carrying out the substitution 336:Gly->Asp to glgC has been reported to increase the yield of glycogen due to the loss allosteric inhibition. This mutated form of glgC is one of the BioBricks which we are in the process of making.
The second phase is the conversion of glycogen to starch. To achieve this we are creating isoamylase and granule-bound starch synthesase BioBricks (isa1, isa2 and gbss) from Zea mays cDNAs. These three genes together should be sufficient for the production of starch from glycogen.
3. Beta-carotene synthesis
Vitamin A deficiency results in night-blindness and an impaired immune system. A number of genes from Pantoea ananatis will be added to our bacterial cells, in essence transferring the beta-carotene synthesis pathway of P. ananatis to our chasis organism. These genes are crtE (geranyl diphosphate synthesase), crtB (phytoene synthase), crtI (phytoene desaturase) and crtY (lycopene beta-cyclase). We will also create BioBricks of the E. coli genes dxs and appY, the addition of which should increase the yield of beta-carotene.
System overview
