Dietary sugar-regulated recombinant protein production in the gut

Probiotic bacteria offer great potential for use as therapeutic delivery vehicles due to their health-promoting phenotypes, a long history of safe consumption, and the ability to survive passage through the mammalian gastrointestinal tract. To express recombinant proteins, genes can be under the control of a constitutive promoter on a plasmid. However, especially for proteins that are less tolerated, this places a metabolic burden on the cell which hampers cell yield. One solution is controlled gene expression, in gram-positive probiotics accomplished by two-component regulatory systems. However, these systems require an inducer peptide to induce gene expression, which complicates the application in the gut ecosystem due to unpredictable pharmacokinetics of the induction peptide during gastrointestinal transit. Moreover, from a safety perspective, plasmids harboring genes conferring antibiotic resistance should be avoided. Thus, there is a critical need to develop technological innovations with an inducer for plasmid-independent recombinant protein production that are independent of induction peptides and antibiotic markers. Our long-term goal is to develop the probiotic human gut symbiont Limosilactobacillus reuteri (Lr) as a therapeutic delivery vehicle for use in humans. Our group has pioneered the approach of exploiting prophage-mediated lysis to release and deliver recombinant proteins, which has proven functional in multiple preclinical models. The overall objective of this application is to develop the innovative concept of plasmid-independent and carbohydrate-regulated production of recombinant proteins. Our central hypothesis is that carbohydrate-regulated promoters fused to recombinant interleukin-22 (IL-22) will reduce liver triglycerides in a diet-dependent manner. Our hypothesis has been formulated based on our preliminary data, demonstrating the identification of tightly controlled sugar-inducible promoters. Also, published work from our laboratory demonstrated that Lr engineered to release IL-22 ameliorates liver triglycerides, which means the chosen preclinical model is a viable approach to test our hypothesis. The rationale for the proposed research is that its successful completion will create exciting opportunities to expand the repertoire of dietary sugars and their combinations in the application of tailored gene expression in the gut, which can be expanded to other microbes and disease models. We plan to accomplish the overall objective by addressing the following specific aims: (1) determine the therapeutic efficacy of dietary sugar-regulated recombinant protein production; (2) determine the therapeutic efficacy following in vitro priming of recombinant protein production. The expected outcomes are to have established that dietary sugars can function as an inducing agent for therapeutic production when relying on in vivo or in vitro carbohydrate metabolism. Thus, a positive impact is expected as our work will create previously unexplored opportunities to control (recombinant) protein production in the gut with uses in basic biology, industry, and medicine. Project Number: 1R21AI194541-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jan-Peter van Pijkeren | Institution: UNIVERSITY OF WISCONSIN-MADISON, MADISON, WI | Award Amount: $427,625 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 MGG-F (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19454101