The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida's chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter-GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.

Pseudomonas putida efficiently utilizes many different carbon sources without the formation of byproducts even under conditions of stress. This implies a high degree of flexibility to cope with conditions that require a significantly altered distribution of carbon to either biomass or energy in the form of NADH. In the literature, co‐feeding of the reduced C1 compound formate to Escherichia coli heterologously expressing the NAD+‐dependent formate dehydrogenase of the yeast Candida boidinii was demonstrated to boost various NADH‐demanding applications. Pseudomonas putida as emerging biotechnological workhorse is inherently equipped with an NAD+‐dependent formate dehydrogenase encouraging us to investigate the use of formate and its effect on P. putida’s metabolism. Hence, this study provides a detailed insight into the co‐utilization of formate and glucose by P. putida. Our results show that the addition of formate leads to a high increase in the NADH regeneration rate resulting in a very high biomass yield on glucose. Metabolic flux analysis revealed a significant flux rerouting from catabolism to anabolism. These metabolic insights argue further for P. putida as a host for redox cofactor demanding bioprocesses.

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It is widely assumed that the thalamus is not involved in olfaction. The ventrolateral thalamus is, however, closely connected to the contralateral cerebellum, which is involved in the sense of smell based on findings from functional imaging studies and findings of olfactory deficits in patients with cerebellar disease. We hypothesized that olfactory deficits following lesions of the ventrolateral thalamus may be similar to olfactory deficits following cerebellar lesions. Fifteen patients with a focal thalamic lesion involving the ventrolateral thalamus were examined and compared to 15 patients with a focal cerebellar lesion and 15 healthy controls. A detailed olfactory test ("Sniffin' Sticks") was used to assess different olfactory functions separately for each nostril. In the group of patients with a lesion of the ventrolateral thalamus, an impairment of the odor threshold was found at the ipsilateral nostril, consistent with the unilateral orientation of the olfactory system in the telencephalon. In the group of patients with a cerebellar lesion, an olfactory deficit at the contralesional nostril emerged. In controls, no significant side difference was found. The involvement of the ventrolateral thalamus in olfaction is comparable to that of the cerebellum in respect to odor threshold. Further study is needed to assess if these findings are related to an impairment of an olfactomotor loop. Present evidence for this hypothesis is indirect. Effects were subclinical as none of the patients reported olfactory disturbance. The results suggest that the cerebello-thalamic axis plays an adjuvant role in olfaction.

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