PF-5

Despite their miniscule mass, microgravity has been shown to impose physiological stresses on bacteria, leading to altered gene regulation and metabolic output in unpredictable ways. Changes in metabolic output can ultimately impact the production of bacterial secondary metabolites. While a few studies have explored the changes in bacterial secondary metabolites under simulated or spaceflight microgravity, the specific area of plant-beneficial compound production by bacteria remains largely unexplored in these contexts. In this study, the production of metabolites pyoluteorin, 2,4-diacetylphloroglucinol (DAPG), orfamide A produced by the plant-promoting bacterium Pseudomonas protegens Pf-5, and pyrroloquinoline quinone (PQQ) produced by Burkholderia ambifaria AMMD under simulated microgravity was analyzed. Transcriptomic analysis of the biosynthetic clusters, coupled with targeted metabolomics, revealed significant downregulation of ofaABC and pqqBCDE operons on Day 3 of incubation under simulated microgravity. This downregulation corresponded to reduced levels of orfamide A and pyrroloquinoline quinone, indicating a functional link between transcriptional suppression and decreased metabolite yield. Two complementary genetic engineering approaches were used to increase the production of these compounds: native chromosomal promoter replacement with either endogenous or inducible promoters, or plasmid-based operon overexpression. While the chromosomal strategy yielded modest expression improvements in P. protegens , the plasmid-based system enabled robust expression with a significant increase in pyrroloquinoline production in B. ambifaria . This study provides a first step for engineering these microbes to provide plant-promoting metabolites in microgravity as a mechanism of aiding the cultivation of plants aboard space-faring vehicles.

Bacteria play an essential role in supporting plant health by producing antifungal compounds that protect against fungal diseases. In space, these bacterial functions may be disrupted. This study examined how microgravity affects the production of such compounds in plant beneficial bacteria, Pseudomonas protegens and Burkholderia ambifaria . Under simulated microgravity, both bacteria showed reduced production of some antifungal compounds due to reduced gene expression. To address this, we used genetic strategies to boost the bacteria's ability to produce these compounds despite the stress of microgravity. Our results showed that engineered bacteria could either partially recover gene expression or even enhance production. These findings are important first steps for engineering microbes to not only enhance plant growth for space agriculture but also provide important information for engineering microbes to boost crop production on Earth as well.

Genetic circuit engineering enables new cellular functions, yet most circuits are developed in the model host Escherichia coli, limiting their availability and performance in alternative organisms. To expand chassis options, we developed an experimental-theoretical pipeline to evaluate NOT logic circuits, or inverters, in the soil bacterium Pseudomonas protegens Pf-5, a species with valuable environmental traits and a newcomer to bioengineering. We characterized the inverter's input-output behavior and used mathematical modeling to infer key dynamic principles. The model quantified how parameters such as translation efficiency, repressor performance, and promoter activity shape circuit output and influence inter-host portability. Pf-5 displayed distinct properties, including steeper on/off transitions than the synthetic biology workhorse Pseudomonas putida. A model-guided design of two compatible inverters connected in series was validated, producing a YES logic response. This work provides DNA parts, circuits, and models that establish P. protegens Pf-5 as a promising chassis for environmental synthetic biology. A record of this paper's transparent peer review process is included in the supplemental information.