Team:Imperial/Project Background


Imperial iGEM 2014



Cellulose is the most abundant organic polymer found in nature. Due to its versatility and ubiquity we find cellulose has applications in areas from medicine to textiles. Much of the cellulose we use is impure as it is derived from plants. Bacteria offer an alternative means of production that produces a cellulose that is purer and requires less processing. In our project we optimise the production of bacterial cellulose (BC) by engineering Gluconacetobacter xylinus and transferring the system into Escherichia coli. We also functionalise our cellulose in order to expand its mechanical, chemical and biological properties into new areas of use.

Bacterial Cellulose

Figure 1: Bacterial cellulose fibrils. (Source: Lee et al. 2014)

A Better Cellulose...

Bacterial cellulose (BC) has the same molecular formula as plant cellulose but is not contaminated by other cell wall components such as lignin and hemicelluloses. This means that its processing is simple not requiring energy or chemicals to isolate the cellulose. This results in a greater degree of polymerisation and a more crystalline structure (Nishi et al. 1990).

With Many Applications...

Bacterial cellulose possesses useful mechanical properties that make it a useful material in many industries. Applications range from biomedical applications in wound dressings and tissue scaffolds (Gama et al. 2013) to high quality papers and diaphragms for high-performance speakers (Nishi et al. 1990). Its use as a filtration material has also been previously investigated (Chen et al. 2009) as well as an adsorbent for metal ions (Oshima et al. 2008).

Produced by Bacteria...

Bacterial cellulose is known to be produced by several species, including Agrobacterium tumefaciens, species from the genus Acetobacter, and in very limited quantities, also in Escherichia coli (Monteiro et al., 2009; Setyawati et al, 2007; Keshk, 2014).

However, one of the highest producing species is Gluconacetobacter xylinus (previously classified as Acetobacter xylinum) ( Lee et al., 2014). Gluconacetobacter species produce cellulose by synthesizing and extruding cellulose polymers through pores in the outer membrane, which aggregate into fibrils and larger ribbons (see Figure 1).

BC Synthesis Pathway

Bacterial cellulose is a biomaterial composed of long, branched, tangled fibrils, each of which consists of intertwined strands of polymerised glucose. Because glucose is the sole building block, cellulose biosynthesis is intimately linked to core carbon metabolism – glucose catabolism, gluconeogenesis, the tricarboxylic acid cycle (TCA cycle) and the pentose phosphate pathway (PPP). In G. xylinus glucose is broken into pyruvate via the Entner-Doudoroff pathway, an alternative to glycolysis with half the energy output (1 ATP and 1 NADH instead of 2 ATP and 2 NADH per glucose) and so the TCA cycle and oxidative phosphorylation are vital for ATP synthesis. G. xylinus is therefore an obligate aerobe, needing oxygen to survive.

Live G. xylinus cells grown in static culture are almost exclusively found in the oxygen-rich upper millimetre of the medium. The species has evolved to find and fit this niche – when oxygen is low the cells down-regulate cellulose synthesis and undergo aerokinesis towards more hospitable environments. When oxygen is abundant they up-regulate the cellulose synthesis pathway and enter a collective biofilm-based mode of survival.

Cellulose can be synthesised from any carbon substrate that can be converted to the precursor glucose 6-phosphate, such as other sugars (e.g. fructose) or smaller organic molecules (e.g. glycerol), but glucose itself is the closest such substrate in G. xylinus metabolic network. The pathway from glucose to cellulose is brief, but intersects two key areas of core carbon metabolism – glucose catabolism and the PPP – and has important implications for a third – the TCA cycle. The pathway consists of 4 steps:

  1. Glucose + ATP → Glucose 6-phosphate + ADP, catalysed by hexokinase or glucokinase
  2. Glucose 6-phosphate ↔ Glucose 1-phosphate, catalysed by phosphoglucomutase
  3. Glucose 1-phosphate +UTP → UDP-glucose + PPi, catalysed by UTP glucose 1-phosphate uridylyltransferase
  4. UDP-glucose + [beta]1-4[Glucose]n → UDP + [beta]1-4[Glucose]n+1, catalysed by cellulose synthase

Reaction 1 is a standard reaction used to convert absorbed glucose into a form that cannot easily leave the cell. Reaction 2 transfers the phosphate to carbon-1 in preparation for reaction 3. Reaction 3 'activates' the glucose by attaching the high-energy pyrophosphate group (bound to deoxyribouridine). Reaction 4 uses the energy stored in the pyrophosphate group to bond the new glucose monomer to the existing polysaccharide chain. Reactions 2, 3 and 4 are the cellulose synthesis pathway, which diverts glucose away from sugar metabolism and ATP synthesis – therefore, in addition to the energy costs of enzyme synthesis, UDP-glucose synthesis and UTP recycling (via UDP) there is a substantial opportunity cost in terms of lost ATP generation as glucose is used to produce an inactive polymer rather than sent to the TCA cycle.


  1. Monteiro, C., Saxena, I., Wang, X., Kader, A., et al. (2009) Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences. Environmental microbiology. [Online] 11 (5), 1105–1116. Available from: doi:10.1111/j.1462-2920.2008.01840.x [Accessed: 31 August 2014].
  2. Setyawati, M.I., Chien, L.-J. & Lee, C.-K. (2007) Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O(2) tension maximizes bacterial cellulose pellicle production. Journal of biotechnology. [Online] 132 (1), 38–43. Available from: doi:10.1016/j.jbiotec.2007.08.012 [Accessed: 5 August 2014]
  3. Keshk, S.M. (2014) Bacterial Cellulose Production and its Industrial Applications. Journal of Bioprocessing & Biotechniques. [Online] 4 (2). Available from: [Accessed: 31 August 2014].
  4. M. Gama, P. Gatenholm, D. Klemm, ‘‘Bacterial Nanocellulose: A Sophisticated Multifunctional Material’’, CRC Press, Boca Raton, FL 2013
  5. Y. Nishi, M. Uryu, S. Yamanaka, K. Watanabe, N. Kitamura, M. Iguchi, S. Mitsuhashi, J. Mater. Sci. 1990, 25, 2997