Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida.

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual- inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modi ﬁ ed for use in P. putida . This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida . Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters P tac or P tetR/tetA . Functional validation of pRGPDuo2 was con- ﬁ rmed by the co-expression of genes for the ﬂ uorescent proteins namely, superfolder green ﬂ uorescent protein (sfGFP), and red ﬂ uorescent protein (RFP). Moreover, the strength of the ﬂ uorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression pro ﬁ ling and adds to the tools available for P. putida metabolic engineering.


Introduction
Pseudomonas putida is a Gram-negative, rod-shaped, soil bacterium that has been widely employed for bio-industrial applications (Tiso et al., 2014). P. putida is considered to be a favorable host for the production of heterologous proteins due to its advantageous traits such as low nutritional requirements and diverse aerobic metabolism (Timmis, 2002). Moreover, physiological features like its ability to generate high biomass yield, rapid growth rate, and minimal maintenance requirements allow P. putida to be developed as an industrial producer for the production of targeted recombinant proteins (Poblete-Castro et al., 2012). Additionally, compared to other bacterial systems, P. putida is known to tolerate various stresses such as toluene, styrene, octanol (Domínguez-Cuevas et al., 2006;Blank et al., 2008) as well as reduced water activity (Hallsworth et al., 2003), adding to its potential as a host to be employed for bioremediation applications and the production of toxic compounds (Kuepper et al., 2015), that would be challenging in bacterial hosts like E. coli and Bacillus spp.
For P. putida to achieve its full potential and to be used as a major microbial host in genetic and biotechnological studies, the key requirement is to develop robust synthetic biology tools for genetic manipulations including plasmid vectors for expression of heterologous proteins and gene knockouts or repression. Over the years, there have been remarkable advances to broaden the existing repertoire of molecular toolbox for P. putida by developing improved and more advanced genome engineering tools for functional analysis or gene expression of heterologous pathways (Martínez-García and de Lorenzo, 2017;Nikel and de Lorenzo, 2018;Martínez-García and de Lorenzo, 2019;Calero and Nikel, 2019). For instance, a series of plasmid vectors are available to use in gram-negative bacteria (including Pseudomonas putida) from Standard European Vector Architecture Database (SEVA) repository that allows a simple exchange of particular module (eg., origins of replication and antibiotic selection) with another vector element (Silva-Rocha et al., 2013). In a recent advancement, the relatively new CRISPR/(d)Cas9 technology has been adapted to manipulate genome or achieve titrable gene expression in P. putida (Tan et al., 2018;Batianis et al., 2020;Wirth et al., 2020). However, it is noteworthy that there is still a limited number of vectors available for this organism that allows the tightly controlled or tunable expression of genes, and that in turn makes rewiring of metabolic pathways difficult .
Most of these vectors harbor a single multiple cloning site downstream of either a constitutive or an inducible promoter system such as P tac , P tet , P BAD . Such vectors are useful but inefficient for cloning large DNA fragments or multiple genes (Radmacher et al., 2002). In these situations, a large number of genes are co-expressed using two or more vectors, each carrying a different antibiotic resistance, therefore alleviating a great deal of stress on bacterial growth (Gauttam et al., 2019b). Especially, for gene co-expression and relieving antibiotic stress, duet-expression vectors have been constructed to allow transcription from individual promoters (Goldbeck and Seibold, 2018;Gauttam et al., 2019a). For instance, the development of a series of T7 promoter based (IPTG-inducible) compatible duet expression vectors in E. coli, P trc -lacI based duo vector in P. putida (Yu et al., 2018) and pDUO duet-expression vector series in Pseudomonas fluorescens (Nakata, 2017) greatly accelerated the co-expression of large number of genes encoding the rate-limiting enzymes in biosynthetic pathways (Tolia and Joshua-Tor, 2006). However, E. coli (and P. putida) duet vectors are IPTG-inducible only, and P. fluorescens duet vectors are arabinose-inducible only, therefore allowing co-expression of two genes but not differential regulation of two genes. Dual-inducible duet-expression vectors (pRG_Duet1, pRG_Duet2 and pRGP_dCas9) have been developed and shown to be functional in Corynebacterium glutamicum (Gauttam et al., 2019a), with each vector harboring two independently inducible promoters, P tac and P tetR/tetA , to regulate the expression of two sets of genes independent of each other (Gauttam et al., 2019a(Gauttam et al., , 2019b. In this study, we have developed a dual-inducible duet expression vector pRGPDuo2 for the expression of heterologous proteins in P. putida. The E. coli -P. putida shuttle expression vector pRGPDuo2 is based on the colE1 replicon of E. coli and the pRO1600 replicon of P. putida, carrying the two multiple cloning sites, MCS1 downstream of P tac promoter and MCS2 downstream of P tetR/tetA promoter.

Bacterial strains, plasmids and culture conditions
Bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were routinely grown in LB medium (Green and Sambrook, 2012) at 200 rpm and 37°C. Unless otherwise stated, P. putida (pre-cultures only) were grown in LB medium at 200 rpm and 30°C. For the cultivation of P. putida, a single colony was picked from a freshly prepared agar plate to inoculate 5 ml of LB medium (first preculture) and incubated for 8 h at 200 rpm and 30°C. The first preculture was used as starter culture for second pre-culture, to inoculate M9 minimal medium (6 g l −1 (Na 2 HPO 4 ), 3 g l −1 KH 2 PO 4 , 1.4 g l −1 (NH 4 ) 2 SO 4 , 0.5 g l −1 NaCl, 0.2 g l −1 MgSO 4 .7H 2 O) with an added 2.5 ml l −1 of a trace elements solution and 0.5% glucose as sole carbon source. The cultivation of all P. putida strains was performed two times in M9 medium before inoculating the main culture for growth and measurement of fluorescence. Bacterial cultures were supplemented with kanamycin (50 μg/ml), carbenicillin (100 μg/ml), IPTG (0 to 1 mM) and/or aTc (0 to 1000 ng/ml), when required. Growth was determined spectrophotometrically by measuring the absorbance at 600 nm (OD 600 ). The relevant information regarding all the strains and plasmids have been deposited in the public instance of the JBEI Registry (http://public-registry.jbei.org/).

DNA preparation, manipulation and transformation
Standard molecular biology procedures were used for DNA isolation, electrophoresis, gene cloning, E. coli competent cells preparation and transformation (Green and Sambrook, 2012). The recombinant strains were selected on LB agar plates (15 g l −1 agar) containing respective antibiotics. Restriction enzymes, T4 DNA Ligase, and alkaline phosphatase employed in this study were obtained from Thermo Fisher Scientific (Waltham MA, USA) and used per instructions from the manufacturer. Oligonucleotides were ordered from Integrated DNA Technologies (IDT, San Diego, California, USA) and are listed in Table 2. The cloned region was sequence-verified by a Sanger Sequencing service (Genewiz, Seattle, WA, USA). Polymerase chain reaction (PCR) conditions were optimized for each primer pair, and DNA fragments were amplified using Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). PCR products were separated by electrophoresis on agarose gels (1% w/v) and purified using the NucleoSpin DNA extraction kit from Qiagen (Hilden, Germany). The recombinant plasmids were isolated from E. coli transformants using the NucleoSpin plasmid purification kit from Qiagen, following the instructions of the manufacturer. P. putida transformations were carried out by electroporation. Briefly, the Pseudomonas strains grown overnight on LB agar plates were resuspended in 300 mM sucrose solution and washed twice before resuspension in 500 μl of 300 mM sucrose. Prior to electroporation, 100 μl aliquots were mixed with 500 ng of the plasmid DNA in a 2-mm cuvette and cells were electroporated using a Gene Pulser XCell™ (BioRad Labs GmbH, Munich, Germany) with parameters set to voltage 2.5 kV, the capacitance of 25 μF and resistance of 200 Ω. Cells were then incubated in LB medium for 2 h for recovery before being plated on selection plates. Basic bioinformatics tools and software were used for designing oligonucleotides such as SnapGene software (GSL Biotech; available at snapgene.com), vector map generation (SnapGene) and genome analysis (NCBI Blast).

Cloning of fluorescent genes (sfGFP and RFP) to construct pRGPDuo2 derived reporter plasmids
Two reporter proteins, a superfolding variant of GFP (sfGFP), and RFP were chosen to validate the functionality of the expression vector, pRGPDuo2. The gene encoding either sfGFP or RFP was cloned in pRGPDuo2 vector in different combination to create following plasmids: pRGPDuo2-sfGFP tac , pRGPDuo2-sfGFP tet , pRGPDuo2RFP tac , pRGPDuo2RFP tet and pRGPDuo2-RFP tac + sfGFP tet . The gene encoding sfGFP was amplified using pGEM00003 as a template and duosfGFPfwd/rev as primers with incorporated restriction sites and subcloned into a cloning vector pJET1.2/blunt, to construct donor plasmid pJET-sfGFP, following the instructions from the manufacturer. Similarly, two variants of RFP donor vectors, namely pJET-RFP tac and pJET-RFP tet , were created using pBADTrfp as a template using duoRFPtac-fwd/rev and duoRFPtet-fwd/rev primers, respectively. These donor vectors were employed to create test plasmids. For instance, plasmid pRGPDuo2-sfGFP tac was constructed by ligating PstI/BamHI-digested pRGPDuo2 with PstI/BamHI-digested PCR product (containing sfGFP DNA sequence) from pJET-sfGFP and plasmid pRGPDuo2-sfGFP tet by ligating NheI/BglII-digested pRGPDuo2 with NheI/BglII-digested product from pJET-sfGFP. The plasmids expressing gene coding for RFP, plasmid pRGPDuo2-RFP tac , was constructed by ligating PstI/BamHI-digested pRGPDuo2 with PstI/BamHI-digested PCR product (containing RFP DNA sequence) from pJET-RFP tac and plasmid pRGPDuo2-RFP tet was constructed by ligating NheI/BglII-digested pRGPDuo2 with NheI/BglIIdigested product from pJET-RFP tet . The final plasmid pRGPDuo2-RFP tac + sfGFP tet for co-expressing gene for both fluorescent proteins (sfGFP and RFP) was created by ligating NheI/BglII-digested pRGPDuo2-RFP tac with NheI/BglII-digested product from pJET-sfGFP.

Fluorescence spectroscopy
The GFP and RFP fluorescence relative to the optical density (OD 600 ) were measured using TECAN infinite M200 plate reader (Mannedorf, Switzerland) using 48 well plates (Sarstedt, Germany) with each well containing 250 μL of cell culture. Cells were induced at 0 h with the appropriate amount of the inducers IPTG and/or aTc. After induction, cells were grown at 30°C for 24 h until fluorescence was measured. For sfGFP, λ ex = 485 nm and λ em = 535 nm were used; and for RFP λ ex = 535 nm and λ em = 620 nm were used. The wavelength for fluorescence excitation and emission were carefully selected to have a little overlap of fluorescence excitation and emission spectra between sfGFP and RFP. Optical density was measured as absorbance at 600 nm. Cell culture fluorescence for each test strain was normalized by its cell density (OD 600 ). All the represented values were mean from at least three independent measurements.

Accession number
The complete nucleotide sequence of plasmid pRGPDuo2 has been submitted to the NCBI GenBank with the accession number MN913428.

Results and discussion
3.1. Construction of a dual-inducible duet-expression shuttle vector pRGPDuo2 in P. putida While vectors for gene co-expression have been developed in E. coli, C. glutamicum, and P. fluorescens, they have not been developed for P. putida. To overcome the shortcomings of the current P. putida expression vectors and to expand the available repertoire of P. putida expression vectors, a duet-expression vector for P. putida, pRGPDuo2, was designed. Two different inducible repressor systems in a single-plasmid system, lacI dependent-repression system controlled by P tac promoter and tetR dependent-repression system controlled by P tetR/tetA were combined to establish tight control over targeted gene expression for metabolic engineering purposes. The previously created duet-expression vector pRGPDuo2 (Gauttam et al., 2019a(Gauttam et al., , 2019b was selected as the backbone to retain all functional elements such as two different multiple cloning sites (MCS1 and MCS2) each controlled by either IPTG-inducible P tac or by aTc-inducible P tetR/tetA promoters, kanamycin resistance gene (Kan R ), transcriptional terminators and the E. coli origin of replication colE1. To adapt the C. glutamicum expression vector pRG_Duet2 for use in P. putida, the Corynebacterium origin of replication (oriV C. glut ) was exchanged with the Pseudomonas origin of replication (Ori P. putida ). To achieve this, the DNA fragment containing P. putida This study a Strain name in bracket corresponds to the part ID assigned to each strain for JBEI public registry.

Table 2
Oligonucleotides used in this study.
When GFP/RFP fluorescence was measured in recombinant strain PP6, the GFP (983 ± 91) and RFP (344 ± 16) fluorescence was found to be significantly increased in this strain compared to the control strain PP1 ( Fig. 2A, B). When GFP fluorescence was measured in P. putida strain PP2 supplemented with IPTG concentrations ranging between 0 and 1000 μM (and a constant aTc concentration of 1000 ng/ml), a steady increase in GFP fluorescence was observed from 86 ± 5 to 998 ± 40 (Fig. 3A). Similarly, in P. putida strain PP3, supplemented with aTc concentrations between 0 and 1000 ng/ml (and a constant IPTG concentration of 1000 μM) a steady increase in GFP fluorescence was observed from 344 ± 28 to 1248 ± 55 (Fig. 3B). The experiment also indicated that the presence of opposite inducer (aTc for P tac and Fig. 1. Plasmid map of shuttle expression vector pRGPDuo2. The dual-inducible duet vector harbors origins of replication for stable maintenance in E. coli (colE1) and P.putida (pRO1600), two multiple cloning sites (MCS1 and MCS2) with sequence for unique restriction enzymes, antibiotic resistance marker (kanamycin), transcriptional terminators, and two independently inducible repression systems, lacI and tetR, controlling the gene expression through MCS1 and MCS2, respectively. MCS1 contains the recognition sequences for the restriction enzymes PstI, SbfI, SalI, BamHI, Acc65I, KpnI, Eco53kI, SacI, and BsaI. MCS2 contains the recognition sequence for BglII, BstZ17I, BmtI, NheI, AvrII, SwaI, BsrGI, BstBI, and SpeI. Abbreviations are as follows: Kan R : kanamycin resistance; tetR: tetracycline repressor; P tetR/tetA : overlapping bacterial promoters of tetR and tetA (aTc-inducible); P tac : tac promoter (IPTG-inducible); lacI q : repressor lacI q ; P lacIq : promoter P lacIq ; terminator: transcriptional terminator. Fig. 2. pRGPDuo2 based expression of GFP and RFP in P. putida KT2440. The following recombinant P. putida strains were investigated for GFP (A) and RFP (B) fluorescence: PP1 (KT2440 carrying pRGPDuo2), PP2 (KT2440 carrying pRGPDuo2-sfGFP tac ), PP3 (KT2440 carrying pRGPDuo2-sfGFP tet ), PP4 (KT2440 carrying pRGPDuo2-RFP tac ), PP5 (KT2440 carrying pRGPDuo2-RFP tet ) and PP6 (KT2440 carrying pRGPDuo2-RFP tac + sfGFP tet ). Data represent mean values of triplicate assays from at least three individual cultivations, and error bars represent standard deviations. IPTG for P tetR/tetA ) does not influence genes from non-cognate promoter. The fluorescence intensities were determined for P. putida PP6, in the presence of different concentrations (ranging between 0 and 1000 μM or ng/ml) of both the inducers (IPTG and aTc) to show the differential expression of sfGFP and RFP present on the same plasmid ( Fig. 3C and D). The gradual increase in both GFP and RFP expression was observed corresponding to a gradual increase in respective inducer(s) concentration ( Fig. 3C and D). Our results provide evidence that the expression of two genes can be regulated independently to one another by adding a single inducer, either IPTG or aTc.

Conclusion
The presented work successfully demonstrates the functionality of dual-inducible shuttle expression vector pRGPDuo1. The constructed vector has two distinct multiple cloning sites (MCS1 and MCS2) controlled by two different controllable repressor systems (lacI and tetR). The fluorescence expression at different inducer doses was tested that showed gradual expression of a target gene, which in turn correlates to the protein level in cells. Such a system can be a promising tool for providing valuable information regarding the expression profile for designing metabolic engineering strategies. The use of single plasmid pRGPDuo2 to differentially regulate the expression of two genes is also an upgrade to the existing double plasmid-based expression system. The use of pRGPDuo2 is particularly promising in situations where differential expression of genes is the key requirement as in the case of cochaperone expression to promote expression of targeted genes (Mueller et al., 2018). The vector pRGPDuo2 will be very useful for gene coexpression studies in P. putida and expands the molecular repertoire of molecular tools for genetic manipulation in this organism.

Ethics approval and consent to participate
Not applicable.

Funding
This work was part of the Department of Energy (DOE) Joint BioEnergy Institute (http://www.jbei.org) supported by the U.S. DOE, Office of Science, Office of Biological and Environmental Research, through Contract No. DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. DOE. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purpose.

Declaration of Competing Interest
The authors declare that they have no conflicts of interest.