A timed off-switch for dynamic control of gene expression in Corynebacterium glutamicum

Introduction
Dynamic control of gene expression mainly relies on inducible systems which require supplementation of (costly) inducer molecules such as IPTG. In contrast, synthetic regulatory circuits which allow the timed shutdown of gene expression are rarely available and therefore represent highly attractive tools for metabolic engineering. Corynebacterium glutamicum uses the cheap and lignin derived compounds ferulic acid, vanillin and vanillic acid as carbon and energy source, which are metabolized towards protocatechuate and subsequently entering the central metabolism (Brinkrolf K et al., 2006). Expression of the vanABK operon, which is required for vanillic acid utilization, is activated by the transcriptional regulator VanR in the presence of vanillic acid, but in vitro not by the pathway precursors ferulic acid and vanillin (Heravi KM et al., 2014). However, in vivo the latter two are degraded to vanillic acid which then acts as the effector molecule for VanR. Since all of these three compounds are co-metabolized with e.g. glucose, this regulatory circuit represents an elegant system to turn-off gene expression when vanillic acid, ferulic acid or vanillin are depleted.
Methods and Results
The pyruvate dehydrogenase complex (PDHC) is essential for growth of C. glutamicum on glucose and was previously shown to be an attractive target to improve pyruvate availability for the production of e.g. L-valine or isobutanol (Eikmanns and Blombach, 2014). To control expression of the PDHC in a vanillic acid-dependent manner, we replaced the native promoter region of the aceE gene, which encodes the E1p enzyme of the PDHC, by vanR and a modified version of the promoter PvanABK yielding C. glutamicum ΔPaceE::vanR PvanABK*. As expected, when growth ceased due to consumption of vanillic acid, C. glutamicum ΔPaceE::vanR PvanABK* utilized the residual glucose to produce significant amounts of pyruvate, L-alanine and L-valine. A comparable growth phenotype and product pattern was achieved when ferulic acid or vanillin were supplemented to the medium instead of vanillic acid. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different final biomass concentrations of the engineered strain, respectively.
Conclusions
As shown in our study, the regulatory circuit based on vanR and the mutated promoter PvanABK represents a suitable metabolic engineering tool for C. glutamicum to dynamically shutdown gene expression with vanillic acid, vanillin and ferulic acid as cheap and abundant effector molecules.

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