One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus...

Within the field of bioproduction, non-model organisms offer promise as bio-platform candidates. Non-model organisms can possess natural abilities to consume complex feedstocks, produce industrially useful chemicals, and withstand extreme environments that can be ideal for product extraction. However, non-model organisms also come with unique challenges due to lack of characterization. As a consequence, developing synthetic biology tools, predicting growth behavior, and building computational models can be difficult. There have been many advancements that have improved work with non-model organisms to address broad limitations, however each organism can come with unique surprises. Here we share our work in the non-model bacterium Actinobacillus succinognes 130Z, which includes both advancements in synthetic biology toolkit development and pitfalls in unpredictable fermentation behaviors. To develop a synthetic biology "tool kit" for A. succinogenes, information gleaned from a growth study and antibiotic screening was used to characterize 22 promoters which demonstrated a 260-fold range of fluorescence protein expression. The strongest of the promoters was incorporated into an inducible system for tunable gene control in A. succinogenes using the promoter for the lac operon as a template. This system flaunted a 481-fold range of expression and no significant basal expression. These findings were accompanied by unexpected changes in fermentation products characterized by a loss of succinic acid and increase in lactic acid after approximately 10 months in the lab. During evaluation of the fermentation shifts, new tests of the synthetic biology tools in a succinic acid producing strain revealed a significant loss in their functionality. Contamination and mutation were ruled out as causes and further testing is needed to elucidate the driving factors. The significance of this work is to share a successful tool development strategy that could be employed in other non-model species, report on an unfortunate phenomenon that needs addressed for further development of A. succinogenes, and provide a cautionary tale for those undertaking non-model research. In sharing our findings, we seek to provide tools and necessary information for further development of A. succinogenes as a platform for bioproduction of succinic acid and to illustrate the importance of diligent and long-term observation when working with non-model bacteria.