Abstract
Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.
Highlights
MmP1 RNA polymerase (RNAP) cassette was successfully integrated into the genome of P. putida KT2440, which significantly improved the expression level of super-folder green fluorescent protein
Optimization of the Copy Number of the MmP1 RNAP Cassette We investigated the effect of the copy number of MmP1 RNAP cassette integrated into P. putida KT2440 on expression
It was found that MmP1 RNAP was highly specific to the MmP1 promoter in P. putida KT2440 and the expression system was strictly regulated by isopropyl β-D-1-thiogalactopyranoside (IPTG) (Supplementary Figure 2) with 82-fold induction
Summary
Pseudomonas putida KT2440, a non-pathogenic soil bacteria, is an attractive chassis for heterologous expression (Timmis, 2002; Dammeyer et al, 2011; Poblete-Castro et al, 2012; Lieder et al, 2015) due to its rapid growth (Martins Dos Santos et al, 2004), metabolic diversity (Poblete-Castro et al, 2012; Nikel et al, 2014, Nikel et al, 2016), rapid generation ability of nicotinamide adenine dinucleotide (Ebert et al, 2011), the operability of genetic manipulation (Martínez-García and de Lorenzo, 2019) and robustness to extreme environments (Nikel and de Lorenzo, 2018). The T7 RNA polymerase-mediated expression system is not always efficient for some non-model microbial strains (Zhao et al, 2017). MmP1 RNAP cassette was successfully integrated into the genome of P. putida KT2440, which significantly improved the expression level of super-folder green fluorescent protein (sfGFP). The insertion site and the copy number of MmP1 RNAP cassette were optimized to improve the efficiency of the MmP1 expression system. We successfully obtained the T7-like expression system in P. putida KT2440, which consisted of the MmP1 RNAP integrated strain and corresponding plasmid. Putida KT2440 with T7 RNAP inserted in the optimal site These expression systems were much more efficient than those previously reported in P. putida KT2440. The expression level of difficult-to-express NDHase was successfully enhanced by using the MmP1 expression system in both P. putida KT2440 and P. putida EM42, demonstrating the high efficiency of the expression system
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