A large amount of bio-waste is burnt or deposited in landfill, releasing harmful greenhouse gases. Much of agricultural bio-waste contains plant dry matter, or lignocellulose, approximately half of which is glucose and 18-30% is xylose. Synthetic biologists can engineer E. coli to grow on lignocellulosic biomass and produce clean bio-energy. However, carbon catabolite repression and diauxic growth controls the order in which different carbon sources are metabolised, and so E. coli preferentially utilises glucose over xylose, meaning xylose content in lignocellulose is not utilised to its full potential. We have increased the xylose uptake of E. coli by inducing a yeast-derived XR-XDH pathway in E. coli to provide an alternative route for catabolising xylose to xylulose-5-phosphate (X5P). We also induced phosphoketolase to alleviate the flux of X5P through glycolysis. We envision our improved E. coli strain to serve as a chassis organism that optimises the efficiency of sustainable bio-energy production.
Every year, our world produces vast amounts of bio-waste, such as agricultural waste, food waste, or processed bio-waste. Due to the inherent difficulty in recycling such perishable bio-waste, they are often deposited in landfills or burnt, intensifying pollution and releasing potent gases, such as methane, which contribute to greenhouse gas emissions.
With the world fast approaching a population of 8 billion people, imagine the amount of waste we produce each day!
Much of the agricultural bio-waste contains plant dry matter, or lignocellulose, which consists of cellulose, hemicellulose, and lignin.
Cellulose is a chain of many glucose units, whilst lignin is comprised of various oxygenated phenylpropane units. Hemicellulose is primarily comprised of xylose, which is the second most abundant sugar in lignocellulosic biomass, after glucose.
Synthetic biology has emerged as one potential solution to alleviate the bio-waste issue, with parts that allow bacteria to convert sugars in bio-waste into clean bio-energy or other useful products. The most abundant laboratory strain within synthetic biology is Escherichia coli.
However, E. coli exhibits carbon catabolite repression (CCR) and diauxic growth. This means that given a medium of both glucose and xylose, E. coli will preferentially utilise glucose first, then experience a lag-phase of minimal growth, before utilising xylose as a food source.
Hence, given a lignocellulosic sugar source, a significant portion of it is unused in the initial duration of bacterial growth, and hence the growth rate will be limited with lag-phases. This ultimately manifests as reduced efficiency for these synthetic biology projects.
Our project was advised and guided by researchers from HydGene Renewables, a startup company that was established upon an iGEM project as part of the 2017 Macquarie_Australia iGEM team. Their project, H2ydroGEM, engineered E. coli bacteria to produce hydrogen through photosynthesis and thus demonstrated the potential of synthetic biology in creating clean energy.
Then, we discovered that a significant portion of lignocellulose was not being utilised efficiently by the diauxic growth pattern of E. coli, limiting its rate of growth and hence reducing the efficiency of bio-energy production. Hence, our team pursued research on potential methods of pursuing an improved E. coli strain which would be capable of concurrent glucose-xylose co-utilisation. We came across a figure of all natural metabolic pathways of xylose in various micro-organisms within a scientific paper called Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al., which sparked our design process for improving the efficiency of E. coli’s xylose metabolism.
Firstly, we analysed the current processes that E. coli utilises to metabolise xylose. E. coli makes use of the XI pathway, in which xylose isomerase (XI) converts xylose to xylulose, then xylose kinase (XK) converts xylulose to xylulose-5-phosphate (X5P), which is then further broken down in the pentose phosphate pathway.
Then, we compared the XI pathway with other major xylose metabolic pathways present in different bacteria. We noticed that a similar pattern of metabolism occurred in the XR-XDH pathway of S. cerevisiae cells, which included an extra step of converting xylose to xylitol through xylose reductase (XR), then xylitol into xylulose through xylitol dehydrogenase (XDH). Otherwise, the conversion from xylulose to X5P and onwards was similar between both the XI and XR-XDH pathways.
The introduction of the XR-XDH pathway would provide multiple ways for xylose to be catabolised into X5P, increasing its xylose uptake. Hence, we identified the induction of the S. cerevisiae XR-XDH pathway in E. coli as a viable method to promote increased xylose uptake.
Furthermore, we hypothesised that, with the addition of another xylose pathway, a flux of X5P would be created. To alleviate this flux, we would also introduce the PK pathway by inducing the expression of phosphoketolase (PK) enzymes that convert X5P into glyceraldehyde-3-phosphate (G3P), which then leads to glycolysis.
We were encouraged by the 2021 Jiangnan_China team’s documentation of their improved part (BBa_K3803016) which combined XR, XDH, and XK gene expressions to induce a higher cell growth and production yield. We were further encouraged by various scientific literature articles which sought to tackle the diauxic growth issue through different approaches, ranging from increasing transmembrane transporter rates of XylE and XylFGH, modifying AraE as a xylose transporter, rewiring transporter preferences, and so on. We had not come across any instances where both the XR-XDH pathway and PK pathway were induced in E. coli, and wanted to pursue this combination.
For each gene that expressed a specific enzyme in the process (XYL1 gene for XR, XYL2 gene for XDH, xylB gene for XK, and xpkA gene for PK), we took its protein sequence from a different bacteria, and converted it into codon-optimised E. coli nucleotide sequences. Each of the parts were synthesised individually, then assembled together to form the new pathways.
We envisioned our improved E. coli strain to be combined with other researchers’ synthetic biology solutions that produce bio-energy (e.g. hydrogen, bio-ethanol), increasing their industrial efficiency through enhanced growth rates of the E. coli. This will in turn lead to increased economic viability and profit of bacterial bio-energy production, and promote a circular economy that makes sustainable use of bio-waste from commercial activities, such as farming.
The immediate and direct scientific impact of our project is the improvement of the efficiency of xylose uptake and utilisation within E. coli cells. Consequently, we envision our project contributing to the enhancement of E. coli as a chassis organism for broader uses across the field of synthetic biology. Specifically, we intend for our engineered E. coli to optimise the production of sustainable energy from unused biomass, by improving the conversion of sugars present within lignocellulose into new sustainable products such as clean hydrogen.
Upon proposed implementation, the efficient production of bio-energy from bacteria will have a wide-reaching impact across the local and global agricultural industry. Farmers can capitalise on unused crops that would otherwise be burnt as sources of biofuel, increasing profit and reducing environmental impact. We would encourage a vast range of sustainable synthetic biology projects to utilise our new E. coli strain to tap into the under-used potential of xylose in lignocellulosic biomass.
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