A new approach to whole-scale organ engineering is the seeding of stem cells upon a decellularized scaffold of a desired organ. Current efforts seek to use this method for whole-scale organ engineering. Here, we report a novel decellularizationrecellularization approach to engineering a human colonic graft with human enterocytes and xenogeneic scaffold. Porcine colon tissue was decellularized with protein-solubilizing agents and recellularized by incubation with Caco-2. Immunohistochemistry, DNA staining, and SEM were used to study the effects upon the scaffold. Exposure to 2%SDS-0.5%Triton solution for four hours resulted in the best decellularization and preservation of microstructure, while tissues treated with 2%SDS-0.5%Triton and 0.05%Trypsin/EDTA supported the best recellularization. Our method provides a potential strategy for streamlined regeneration of a viable colonic lumen from xenogeneic and allogeneic sources while maintaining colon microstructure and potential for recellularization.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.