(380c) Engineering A Synthetic Microbial Consortium for Efficient Production of Biofuels

Production of liquid biofuels via microbial conversion of cellulosic feedstocks could sustainably replace much of the fossil fuel consumed in the transportation sector, but tremendous research efforts are still needed in engineering microorganisms with the desired properties. Inspired by the widespread occurrence of synergistic microbial communities in nature, we are investigating a promising but under-explored direction for engineering microorganisms for biofuel production: the design and construction of a synthetic microbial consortium, composed of various microorganisms, each specialized for a specific task in the conversion of lignocellulosic biomass to a biofuel. Three microbial specialists are utilized for the consortium: the cellulolytic fungus Trichoderma reesei RUT-C30, which secretes cellulase enzymes to hydrolyze lignocellulose into component hexose and pentose mono/oligosaccharides, an engineered E. coli hexose specialist that ferments hexose hydrolysis products, and an engineered E. coli pentose specialist that ferments pentose products. In contrast to the ?superbug? paradigm of trying to engineer one organism with all the necessary functionalities to produce biofuels from cellulose, our approach can potentially be more tractable from an engineering and feasibility standpoint. In our initial work, we focus on developing the cellulose utilization aspect of the consortium. The modular nature of our system will allow it to be readily adapted to the large portfolio of existing E. coli strains metabolically engineered to produce biofuels or commodity chemicals.

Lignocellulose is highly recalcitrant to biological degradation [1]. Biofuel production hosts such as S. cerevisiae have been engineered to ferment cellulose via heterologous cellulase production, but reported cellulose hydrolysis rates and yields are modest [2]. In contrast, natural saprophytic organisms have evolved highly specialized and sophisticated synergistic enzyme systems for efficient degradation of cellulose [1]. The filamentous fungus Trichoderma reesei RUT-C30 is a well characterized, prodigious cellulase producer [1] and is utilized as a base strain for the cellulolytic specialist in our consortium. Antagonistic interactions between filamentous fungi and bacteria are commonly observed in nature, but we have successfully co-cultured E. coli K12 and T. reesei RUT-C30 on minimal medium with crystalline cellulose (Avicel) as the sole carbon source, demonstrating the feasibility of the consortium concept.

Diauxic sugar utilization in E. coli reduces fermentation efficiency, motivating the development of hexose and pentose specialist E. coli strains [3]. A pentose specialized E. coli was developed by disabling hexose metabolism via a series a gene deletions (ptsG, glk, and manX); presently we are working to improve utilization of pentose sugars, which are metabolized less efficiently than hexose sugars. For the hexose specialist, our efforts are focused on engineering this strain to utilize cellooligosaccharides, which are primary products of enzymatic cellulose hydrolysis and are not natively metabolized by E. coli. The cryptic chbBCARF operon of E. coli encodes a functional cellobiose permease and phospho-β-glucosidase, but is constitutively repressed in most laboratory strains [4]. We have engineered a cellobiose utilizing strain by expressing the chbBCARF operon on a plasmid with a heterologous promoter and are working to further improve cellobiose metabolism.

Preliminary modeling work indicates that the populations of each consortium member must be balanced for maximum biofuel production, and that the optimal consortium composition may vary with the cellulosic feedstock concentrations and makeup. Although the sub-populations can be controlled by varying the inoculation ratio of the consortium members, the system would be much more robust if the organisms could be coordinated and regulated with external signals (e.g. inducers) or even those internal in the consolidated bioreactor (e.g. feedstock composition). To address this need, we are also engineering synthetic metabolic and genetic circuits to allow organisms to sense and respond to population levels and available substrates.

[1] Lynd, Lee R., Willem H. Van Zyl, John E. McBride, and Mark Laser. "Consolidated Bioprocessing of Cellulosic Biomass: an Update." Current Opinion in Biotechnology 16.5 (2005): 577-83.

[2] Wen, Fei, Jie Sun, and Huimin Zhao. "Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol." Applied and Environmental Microbiology 76.4 (2010): 1251-260.

[3] Eiteman, Mark A., Sarah A. Lee, and Elliot Altman. "A Co-fermentation Strategy to Consume Sugar Mixtures Effectively." Journal of Biological Engineering 2.3 (2008).

[4] Kachroo, Aashiq H., Aswani K. Kancherla, Nongmaithem S. Singh, Umesh Varshney, and Subramony Mahadevan. "Mutations That Alter the Regulation of the Chb Operon of Escherichia Coli Allow Utilization of Cellobiose." Molecular Microbiology 66.6 (2007): 1382-395.