Inducible and tunable gene expression systems for Pseudomonas putida KT2440

Chandran Sathesh-Prabu,Sung Kuk Lee,Rameshwar Tiwari,Doyun Kim

doi:10.1038/s41598-021-97550-7

Chandran Sathesh-Prabu, Sung Kuk Lee + Show 2 more

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https://doi.org/10.1038/s41598-021-97550-7

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Abstract

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 in Pseudomonas 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. 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.

Highlights

Inducible and tunable expression systems are essential for the microbial production of biochemicals
To increase the strain applicability, we aimed to develop a set of promoters that could be regulated either by a carbon source or substrate in P. putida KT2440
We focused on evaluating the efficiency of commonly used and naturally abundant carbon sources, including glucose (Glu), levulinic acid (LA), and xylose (Xyl), on the induction of the expression of a green fluorescent protein (GFP) as a quantitative reporter of relative promoter activity

Summary

Introduction

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 in Pseudomonas 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. A variety of native ( Pm, Psal, PalkB, Pu, and PxylA) and heterologous ( ParaB, PrhaB, Ptrc, Plac, Ptac, Ptet, PT7-lac and PlacUV5, PmekA, PmtlE, PchnB, and PDB3) inducible expression systems have been demonstrated in P. putida strains[9,10,11] These induction systems use a wide range of inducing chemicals such as 3-methylbenzoate, 3-methylbenzyl alcohol, dicyclopropylketone, methyl ethyl ketone, m-toluate, salicylate, n-octane, 3-chloro-4-hydroxyphenylacetic acid, cyclohexanone, rhamnose, arabinose, xylose, mannitol, p-cumate, anhydrotetracycline, and isopropyl-β-D-1-thiogalactopyranoside (IPTG) with varying degrees of success[9,10,11,12,13]. The microbial production of industrially relevant biochemicals using Glu- and LA-inducible expression systems has been previously reported[20,21], the promoters have not been characterized in detail

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