Production of cellulosic organic acids via synthetic fungal consortia.

Consolidated bioprocessing (CBP) is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose, and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover (CS) to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/hr. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules.

Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass.

Synergistic microbial communities are ubiquitous in nature and exhibit appealing features, such as sophisticated metabolic capabilities and robustness. This has inspired fast-growing interest in engineering synthetic microbial consortia for biotechnology development. However, there are relatively few reports of their use in real-world applications, and achieving population stability and regulation has proven to be challenging. In this work, we bridge ecology theory with engineering principles to develop robust synthetic fungal-bacterial consortia for efficient biosynthesis of valuable products from lignocellulosic feedstocks. The required biological functions are divided between two specialists: the fungus Trichoderma reesei, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into desired products. We developed and experimentally validated a comprehensive mathematical model for T. reesei/E. coli consortia, providing insights on key determinants of the system's performance. To illustrate the bioprocessing potential of this consortium, we demonstrate direct conversion of microcrystalline cellulose and pretreated corn stover to isobutanol. Without costly nutrient supplementation, we achieved titers up to 1.88 g/L and yields up to 62% of theoretical maximum. In addition, we show that cooperator-cheater dynamics within T. reesei/E. coli consortia lead to stable population equilibria and provide a mechanism for tuning composition. Although we offer isobutanol production as a proof-of-concept application, our modular system could be readily adapted for production of many other valuable biochemicals.

Microbial utilization of aqueous co-products from hydrothermal liquefaction of microalgae Nannochloropsis oculata.

Hydrothermal liquefaction of algae biomass is a promising technology for the production of sustainable biofuels, but the non-oil, aqueous co-product of the process has only been examined to a limited extent. The aqueous phase from liquefaction of the alga Nannochloropsis oculata (AqAl) was used to make growth media for model heterotrophic microorganisms Escherichia coli, Pseudomonas putida, and Saccharomyces cerevisiae. Growth rates, yields, and carbon/nitrogen/phosphorus uptake were measured. E. coli and P. putida could grow using AqAl as the sole C, N, and P source in media containing 10 vol.%-40 vol.% AqAl with the best growth occurring at 20 vol.%. S. cerevisiae could grow under these conditions only if the media were supplemented with glucose. The results indicate that in a biorefinery utilizing algae liquefaction, the aqueous co-product may be recycled via microbial cultures with significantly less dilution than previously published methods.

Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

Network benchmarking: a happy marriage between systems and synthetic biology.

In their new Cell paper, Cantone et al. (2009) present exciting results on constructing and utilizing a small synthetic gene regulatory network in yeast that draws from two rapidly developing fields of systems and synthetic biology.