Inducible and tunable gene expression systems for Pseudomonas putida KT2440

Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated inPseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/PhpdH was also efficiently induced by LA. LvaR/PlvaA and XutR/PxutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/Pzwf1 showed weak GFP expression.

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Fig: Schematic representations of the activation of the substrate-inducible promoter systems. (a) activation of the HexR/Pzwf1 system by glucose through the EDP effector molecule KDPG; (b) activation of the LvaR/PlvaA system by LA or 4HV; (c) activation of the HpdR/PhpdH system by 3HP or LA; (d) activation of the MmsR/PmmsA system by 3HP or LA; (e) activation of the XutR/PxutA system by xylose. The numerical values (mean ± standard deviation) and the inducer(s) represent the maximum normalized fluorescence intensity of the corresponding system with the respective inducer(s). Both 3HP and LA (3HP/LA) showed the maximum level of expression (P > 0.05) in (c) and (d). EDP, Entner–Doudoroff pathway; KDPG, 2-keto-3-deoxy-6-phosphogluconate; gfp, green fluorescent protein; Glu, glucose; LA, levulinic acid; 4HV, 4-hydroxyvalerate; 3HP, 3-hydroxypropionic acid; Xyl, xylose.

These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.

Sathesh-Prabu, C., Tiwari, R., Kim, D. et al. Inducible and tunable gene expression systems for Pseudomonas putida KT2440. Sci Rep 11,18079 (2021). https://doi.org/10.1038/s41598-021-97550-7

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