Biotechnological production of platform chemicals through anode assisted fermentation by using an artificial biofilm of S. oneidensis

Abstract

A shift from petrochemical processes to a bio-based economy is inevitable to establish a sustainable industry. Bioelectrochemical systems (BESs) are a future technology for the environment-friendly production of platform chemicals. In BESs exoelectrogenic bacteria such as Shewanella oneidensis can directly transfer respiratory electrons to the anode, which serves as a non-depletable electron acceptor. So far, the main limiting factor in BESs is the achievable current density which correlates to some extend with the density, thickness and metabolic activity of anode biofilms composed of exoelectrogenic microorganisms. This is especially true for S. oneidensis as the organism forms rather thin biofilms under anoxic conditions on anode surfaces.

In order to enhance the organisms’ biofilm formation capabilities Bursac et al. deleted the λ-prophage from the genome. The deletion of the λ-prophage led to a 2.3-fold increased cell number on the anode ongoing with a 1.34-fold increased mean current density (Bursac et al., 2017). Furthermore, we just recently discovered that exogenous riboflavin enhances biofilm formation by the upregulation of the Ornithine-decarboxylase speC. This is probably based on a quorum sensing effect of riboflavin. Taken together the upregulation of speC ongoing with the deletion of the λ-prophage leads to a 4-fold increase in current density ongoing with a 6.1-fold increased biofilm formation on the anode.

However, to ensure an optimal performance of the biofilm in BESs, biofilm thickness itself is not sufficient. The biofilm also needs to be conductive. Our aim is to establish the Spytag-/Spycatcher-tool to synthetically steer biofilm conductivity. Spytag and Spycatcher are two protein residues from the fibronectin binding protein of Streptococcus pyogenes (Spy). These two protein residues form a spontaneous isopeptide bond under a variety of temperatures, pH values and buffers (Zakeri et al., 2012). By coupling Spytag and Spyctacher to different outer membrane c-type cytochromes of S. oneidensis the cells are covalently bound to each other while the biofilm remains conductive. In a first application the production of acetoin as one of the top 30 platform chemicals world-wide is desired (US Department of Energy, 2004).

In order to render S. oneidensis producing acetoin instead of the native end product acetate, Bursac et al. deleted the key genes for acetate production and introduced the acteoin production pathway (Bursac et al., 2017). To broaden the substrate spectrum of S. oneidensis further genes for glucose metabolism were introduced. Through a long term adaption, the glucose degradation, the biofilm formation abilities and the bioelectrochemical performance were significantly enhanced.

Merging all genetic optimizations into one production strain will enable us to produce acetoin from glucose as a platform chemical with high space-time yields. This will give rise to a production process that is competitive with existing oxic process routines without being dependent on expensive aeration.

References:

Bursac, T., Gralnick, J.A.,Gescher, J. (2017) Acetoin production via unbalanced fermentation in Shewanella oneidensis. Biotechnol Bioeng 114: 1283–1289.

Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., Howarth, M. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109: E690.