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Gluconeogenesis, the reciprocal pathway of glycolysis, is an energy-consuming process that generates glycolytic intermediates from non-carbohydrate sources. In this study, we demonstrate that robust and efficient gluconeogenesis in bacteria relies on the allosteric inactivation of pyruvate kinase, the enzyme responsible for the irreversible final step of glycolysis. Using the model bacterium Bacillus subtilis as an example, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis via its extra C-terminal domain (ECTD), which is essential for autoinhibition and metabolic regulation. Physiologically, a B. subtilis mutant lacking the ECTD in pyruvate kinase displayed multiple defects under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects were not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two major metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: failure to establish high phosphoenolpyruvate (PEP) concentrations necessary for robust gluconeogenesis and increased carbon overflow into the medium. In silico analysis revealed that, in wild-type cells, an expanded PEP pool enabled by pyruvate kinase inactivation is critical for maintaining the thermodynamic feasibility of gluconeogenesis. Additionally, we discovered that B. subtilis exhibits glyphosate resistance specifically under gluconeogenic conditions, and this resistance depends on the PEP pool expansion resulting from pyruvate kinase inactivation. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient carbon utilization, and antimicrobial resistance.IMPORTANCEPyruvate kinase catalyzes the final irreversible step in glycolysis and is commonly thought to play a critical role in regulating this pathway. In this study, we identified a constitutively active variant of pyruvate kinase, which did not impact glycolysis but instead led to multiple metabolic defects during gluconeogenesis. Contrary to conventional understanding, these defects were not due to the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Our findings suggest that the defects arose from an insufficient buildup of the phosphoenolpyruvate pool and an increase in carbon overflow metabolism. Overall, this study demonstrates the essential role of pyruvate kinase allosteric regulation during gluconeogenesis in maintaining adequate phosphoenolpyruvate levels, which helps prevent overflow metabolism and enhances the thermodynamic favorability of the pathway. This study also provides a novel link between glyphosate resistance and gluconeogenesis., Competing Interests: The authors declare no conflict of interest.

Bacterial biotherapeutic delivery vehicles have the potential to treat a variety of diseases. This approach obviates the need to purify the recombinant effector molecule, allows delivery of therapeutics in situ via oral or intranasal administration, and protects the effector molecule during gastrointestinal transit. Lactic acid bacteria have been broadly developed as therapeutic delivery vehicles though risks associated with the colonization of a genetically modified microorganism have so-far not been addressed. Here, we present an engineered Limosilactobacillus reuteri strain with reduced colonization potential. We applied a dual-recombineering scheme for efficient barcoding and generated mutants in genes encoding five previously characterized and four uncharacterized putative adhesins. Compared with the wild type, none of the mutants were reduced in their ability to survive gastrointestinal transit in mice. CmbA was identified as a key protein in L. reuteri adhesion to HT-29 and enteroid cells. The nonuple mutant, a single strain with all nine genes encoding adhesins inactivated, had reduced capacity to adhere to enteroid monolayers. The nonuple mutant producing murine IFN-β was equally effective as its wild-type counterpart in mitigating radiation toxicity in mice. Thus, this work established a novel therapeutic delivery platform that lays a foundation for its application in other microbial therapeutic delivery candidates and furthers the progress of the L. reuteri delivery system towards human use.IMPORTANCEOne major advantage to leverage gut microbes that have co-evolved with the vertebrate host is that evolution already has taken care of the difficult task to optimize survival within a complex ecosystem. The availability of the ecological niche will support colonization. However, long-term colonization of a recombinant microbe may not be desirable. Therefore, strategies need to be developed to overcome this potential safety concern. In this work, we developed a single strain in which we inactivated the encoding sortase, and eight genes encoding characterized/putative adhesins. Each individual mutant was characterized for growth and adhesion to epithelial cells. On enteroid cells, the nonuple mutant has a reduced adhesion potential compared with the wild-type strain. In a model of total-body irradiation, the nonuple strain engineered to release murine interferon-β performed comparable to a derivative of the wild-type strain that releases interferon-β. This work is an important step toward the application of recombinant L. reuteri in humans., Competing Interests: J.-P.V.P received unrestricted funds from BioGaia AB, a probiotic-producing company. J.-P.V.P is the founder of the consulting company Next-Gen Probiotics, LLC. J.-P.V.P. and L.M.A. are inventors on a patent application describing the development of microbes with reduced adhesion characteristics

Importance: Lactic acid bacteria constitute a genetically diverse group of microorganisms with significant roles in the food industry, biotechnology, agriculture, and medicine. A core understanding of bacterial physiology in diverse environments is crucial to select and develop bacteria for industrial and medical applications. However, there is a lack of versatile tools to track (recombinant) protein production in lactic acid bacteria. In this study, we adapted a peptide-based bioluminescent tagging system that is functional across multiple genera and species. This system enables tracking of tagged proteins both in vitro and in situ , while it also can be used to enumerate recombinant bacteria from the mouse gastrointestinal tract with accuracy comparable to that of conventional plate counts. Our work expands the lactic acid bacteria genetic toolbox and will facilitate researchers in industry and academia with opportunities to monitor microbes and proteins under different physiologically relevant conditions., Competing Interests: Jan-Peter van Pijkeren received unrestricted funds from BioGaia AB, a probiotic-producing company. J.-P.V.P. is the founder of the consulting company Next-Gen Probiotics, LLC.