Engineered probiotics to enhance the production of beneficial bacterial metabolites in the intestine

The gut microbiome interacts intimately with its host and its disruption has been associated with several pathological conditions. The main effectors behind those interactions are part of the microbiome’s metabolic output; the gut bacterial metabolites. Properties such as their natural occurrence, functional pleiotropy, and tissue bioavailability render them excellent candidates for the development of microbiome interventional strategies. As traditional probiotic interventions do not provide consistent therapeutic effects and drug administration in the form of traditional capsules entails therapeutic product loss and increased systemic exposure, an optimal approach can be the development of probiotics specifically engineered to enhance gut bacterial metabolite production localized in the microbiome.

Thus, this project aimed to develop engineered probiotic strains able to produce indole lactic acid (ILA) and valeric acid, two gut metabolites of bacterial origin, and to explore their potential in battling pathological conditions of the host. An ILA-producing strain might assist in treating conditions such as ulcerative colitis that involve intestinal inflammation and a valeric acid-producing strain might be of use against gastrointestinal infections of Clostridium difficile.

The present thesis expands into four chapters. First, an introduction summarizing current knowledge on the gut microbiome and its products, as well as on advanced microbial therapeutic (AMT) approaches. Second, a detailed presentation of the aims of the conducted projects and a summary of the findings. The third chapter comprises all the results. The main findings consist of published or in-preparation manuscripts that are integrated as sections of the third chapter (Manuscript I, Manuscript II), and projects that are not intended for publication are presented in the same chapter as reports (Project I, Project II). The last chapter hosts a discussion based on the presented findings and the gut metabolite-producing AMTs' prospects.

Briefly, an ILA-producing AMT, namely EcN aldh, was successfully developed by the combination of an innate and an extrinsic to Escherichia coli metabolic steps. An aromatic dehydrogenase from Bifidobacterium longum subspecies infantis provided the extrinsic step. EcN aldh was capable of increasing ILA in situ in the murine gut (Manuscript I). Tested on a chemically induced colitis model, the strain showed a mild association with recovery from intestinal inflammation as it affected the expression of several markers of inflammation, but not the macroscopic symptoms of colitis (Manuscript II). To further boost ILA production, we investigated a gene suspected of encoding for an aminotransferase responsible for ILA production in Bifidobacterium longum subs. infantis, although the results were inconclusive (Project I). As the full production pathway of valerate is not yet genetically characterized, a fosmid library construction approach was employed to identify the genes responsible for valerate production (Project II). The generated fosmid clones did not yield any valerate, thus no valerate-producing strain was developed.