These days, the whole bioeconomy is dependent on one valuable resource: sugars. No matter if they are produced from starch or cellulose, they always originate from plants. But while the global demand for carbon sources is growing, the arable land is shrinking and droughts are more frequent.

At iGEM Aachen, we aim to make the bioeconomy independent from plant-derived sugars. We do this by teaching E. coli to use methanol as a carbon source and convert it to glycogen, the bacterial equivalent to starch.

Therefore, we introduce a synthetic pathway, implementing it in vivo for the first time.

For this kind of metabolic engineering research, chemostat cultivation of candidate strains is essential. However, traditional continous cultivations are very costly and not affordable for everyone. To solve this problem, we are developing a do-it-yourself bioreactor with very low culture volume. This bioreactor is accompanied by hardware and software that is cost-effective and user-friendly.

Concering the DIY principle of our modular selfmade bioreactor we established contacts to community labs to discuss about the developement of the European Community Lab Scene.

Regarding biosafety in the lab we tested and discussed our new documentation system with other teams to explore the technology and experiences.

Motivation

When we first started with extensive literature research at the beginning of the project, we came across an interesting paper about an ATP neutral cycle for methanol uptake. ^[1] Right from the beginning we saw the great potential of methanol as a carbon source that is currently produced in large amounts but only partially used for fuels and not nearly up to its full potential. At this point we had the base for our project idea. It was further developed by research on other sources of methanol. We investigated the technical fixation of CO₂ from the air and found out that methanol can easily be produced with this method as it is already by some companies.^[2] But what comes next after methanol uptake and how can we make it available for various industries? The answer is sugars. In the bioeconomy most processes rely on sugar and therefore we decided to convert the methanol into glycogen, the storage molecule of sugar in bacteria. This contributes to making the bioindustry independent of plants and instead rely on renewable methanol.

How We Improved a Previously Existing BioBrick

Enhancing the glycogen accumulation in Escherichia coli is an important aspect of our project. In this context we developed our glycogen synthesis operon (glgCAB). It is a composite part that combines all 3 glycogen formation enzymes. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. The GlgC is based on the part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] from Team Edinburgh 2008, but the RBS B0034 was added to the existing Biobrick. Since we could prove that strains expressing our polycistronic construct accumulate more than strains just expressing glgC, this part is an extension and improvement of the existing Part BBa_K118016.

The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in BL21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector.

SDS-PAGE of glgCAB in pSB1A30 glgCAB in pSB1A30 was expressed in BL21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes . The BL21 Gold (DE3) wild type was used as the negative control.

The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of BL21 Gold (DE3) transformants of BBa_K1585321 indicates considerably more glycogen accumulations compared to the wild type.

Iodine staining BL21 Gold (DE3) + glgCAB vs. wild type Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + glgCAB stained distinctly darker than the BL21 Gold (DE3) wild type.

References

1. ↑ Bogorad IW, Chen CT, Theisen MK, Wu TY, Schlenz AR, Lam AT, Liao JC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15928-33. doi: 10.1073/pnas.1413470111. Epub 2014 Oct 29. PubMed PMID: 25355907; PubMed Central PMCID: PMC4234558.
2. ↑ http://www.sunfire.de/en/