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Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency

High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.


1
Introduction 31 Cell factories have become an established role player in the sustainable production of chemicals and 32 biological products proven with hundreds of billions of USD/year value on global markets (Davy et 33 al., 2017). A commonality in the development of such cell factories is the continuous pursuit of 34 increased productivities through directed or selection-based genetic engineering methods. With both 35 approaches, increasing activity of the partaking pathways commonly leads to the desired rise in 36 productivity. High enzyme activity can be achieved by optimization of transcription, translation, post-37 translational modifications, and the process conditions . A common strategy is to 38 employ strong promoters to overexpress product biosynthesis genes. Highly-active promoters achieve 39 increased protein production rates by increasing the respective mRNA levels in the cell. However, 40 previous studies have shown that high, recombinant gene expression leads to metabolic burden and 41 consequently to growth impairment (Borkowski et al., 2016; Carneiro et al., 2013). Such hindrances 42 are related to the drainage of biosynthetic precursors, such as nucleotides, or seizing of the cellular 43 transcriptional machinery. 44 In recent years, Synthetic Biology parts emerged that support high enzyme activities without the need 45 for strong gene expression, thereby contributing to diminishing competition and depletion of the 46 cellular mRNA pool and lightening the metabolic burden. Two translation-focused approaches can be 47 distinguished that target to optimize translation rather than transcription. To this end, the first approach 48 attempts to stabilize mRNA, whereas the second seeks to increase translational efficiency. Increasing 49 mRNA stability is possible by placing stabilizing sequences in the 5' untranslated region (UTR) that 50 avoid endoribonuclease attacks through their secondary structures as in Escherichia coli (Carrier and 51 Keasling, 1997; Viegas et al., 2018). The implementation of ribozymes upstream of the ribosome 52 binding site (RBS) allows the insulation of the desired expression cassette from the genetic context 53 (Lou et al., 2012). Besides the intrinsic cleaving activity, the ribozymes developed by Lou et al. 54 contained a 23 nucleotide hairpin downstream of the catalytic core, which additionally adds an mRNA 55 stabilizing trait to this genetic part (Clifton et al., 2018). 56 One approach within the second category, which focuses on translational efficiency, allows increased 57 expression levels by facilitating the access of the ribosome to the RBS. In the traditional operon 58 architecture (Figure 1 A), it is possible that gene of interest (GOI)-dependent secondary structures 59 arise. This folding of the mRNA can block the access of ribosomes to the RBS thereby compromising 60 the desired gene expression (Salis et al., 2009). Mutalik et al. developed a 'bicistronic design' which 61 takes advantage of the intrinsic helicase activity of ribosomes to unveil any RBS-GOI dependent 62 secondary structure and consequently achieve GOI-independent expression (Mutalik et al., 2013). In 63 the bicistronic design, a short leading peptide cistron is allocated upstream of the GOI. The RBS of the 64 GOI is enclosed within the coding sequence of the leading cistron, whereas the start codon of the GOI 65 is fused to the stop codon of the leading cistron. The leading RBS-small peptide combination is known 66 to not create any secondary structures, which assures the binding of a ribosome. Once the ribosome 67 binds to the first RBS and starts to translate the leading peptide, any possible downstream RBS-GOI 68 dependent secondary structures are unveiled by its intrinsic helicase activity exposing the RBS of the 69 GOI. Otto et al. recently combined the bicistronic design with an upstream ribozyme to increase the 70 translational efficiency of heterologous genes integrated in rRNA operons. Here the intention of the 71 ribozyme integration was not to stabilize the mRNA but to increase translation efficiency by reducing 72 a potential steric hindrance by the bulky 5' 16S mRNA flank and thereby facilitating ribosome docking. 73 To this end, the ribozyme was placed upstream of the RBS, and this integration indeed resulted in a 74 substantial increase in protein production . 75 Nielsen et al. proposed the compilation of these and further genetic parts into an overall standardized 76 gene expression cassette for the assembly of genetic circuits (Figure 1 B)  proteins (msfGFP and mCherry) and recombinant acetoin production as readout (Landgraf, 2012). 97 The constructed, optimized gene expression cassettes were evaluated on a plasmid basis and after 98 single-copy genomic integration (Wierckx et al., 2005). Overall, the traditional and optimized gene 99 expression cassette variants were characterized with three constitutive and two inducible promoters. 100 Reducing the overall size of the optimized gene expression cassette while maintaining its performance 101 was also targeted in this work. Besides the characterization of several constructs, qPCR analysis was 102 performed to elucidate the role of mRNA stability in altered protein expression. 103 We chose Pseudomonas taiwanensis VLB120 as expression host as this Gram-negative bacterium 104 exhibits industrial relevant, metabolic capabilities such as broad carbon source utilization, the ability 105 to proliferate in the presence of organic solvents, and an almost byproduct free metabolism (Köhler et 106 al., 2013;Park et al., 2007). P. taiwanensis VLB120 has been proven a suitable biocatalyst for the 107 production of (S)-styrene oxide, phenol, isobutyric acid, and 4-hydroxybenzoic acid ( throw of 50 mm in LB medium or LB medium supplemented with 5 g/L glucose and buffered with 115 11.64 g/L K2HPO4 and 4.89 g/L NaH2PO4 (LBmod). Pseudomonas strains were grown at 30°C, whereas 116 E. coli was grown at 37°C. Solid LB was prepared by adding 1.5 % (w/v) agar to the medium. 117 Antibiotics were supplemented to the medium for plasmid maintenance and selection purposes. 118 Kanamycin sulfate was added at a concentration of 50 mg/L for Pseudomonas and E. coli. Gentamycin 119 was used at a concentration of 25 mg/L for both species. Tetracycline was added only to solid media 120 at a concentration of 30 mg/L for Pseudomonas and 10 mg/L for E. coli. To induce the pTN1 derived 121 plasmids harboring the nagR/PnagAa promoter system controlling the expression of the fluorescent 122 proteins or the acetoin pathway, 0.01 mM or 1 mM of sodium salicylate was added, respectively. The 123 inducer isopropyl-β-D-1 thiogalactopyranoside (IPTG) was used at a concentration of 1 mM to induce 124 the Ptrc controlled constructs integrated into the attTn7 site of Pseudomonas strains. 125 The acetoin producing strains were cultivated in airtight 500 mL serum flasks containing 50 mL of 126 LBmod supplemented with gentamycin (see above). The main cultures were inoculated from an 127 overnight pre-culture to an OD600 of 0. Pseudomonas transformants were screened through colony PCR using the OneTaq 2×Master Mix with 159 standard buffer after lysing colony cell material in alkaline polyethylene glycol, as described by 160 Chomczynski and Rymaszewski. (Chomczynski and Rymaszewski, 2006) Successful plasmid 161 constructions and genome integrations were confirmed by Sanger sequencing performed by Eurofins 162 Genomics. All strains and primers used in this work are shown in Table 1. 163

Gravimetric cell dry weight determination 165
Overnight cultures of P. taiwanensis VLB120 were diluted to pre-established optical densities and 166 filtered through membrane filters with a pore size of 0.2 µM which were previously weighted (w0) 167 after being dried in a microwave for 3 min at 350 W. The filter cake was washed three times with 168 distilled water, dried in the microwave for 8 min at 350 W and cooled down in a desiccator before 169 weighing (w1). The cell dry weight (CDW) was calculated by subtracting w0 from w1 and divided by 170 the volume of the filtered suspensions. 190 µL of the same cell suspensions were transferred to a 96-171 well microtiter plate (Greiner Bio One), and scattered light signals were recorded at 620 nm with a 172 gain of 30. Linear regression between the CDW and scattered light signals of the pre-established cell 173 suspensions retrieved the conversion factor between these two units. 174

Fluorescent measurements 175
All Pseudomonas strains expressing fluorescent reporter proteins were characterized in the 176 microbioreactor system BioLector (m2p-labs, Baesweiler, Germany and rpoB were quantified using the linear calibration curves used for primer pairs efficiencies, which 213 were constructed with either the plasmid harboring the traditional msfGFP expression cassette under 214 the control of the SPA75 or genomic DNA, respectively. The data was analyzed with the Bio-Rad CFX 215 Manager and Microsoft Excel software. As we normalized the msfGFP mRNA abundance data with 216 the transcript abundance of the housekeeping gene rpoB, the determination of an absolute decay rate 217 for the msfGFP mRNA was not possible. We calculated the delta between the decay rates of the 218 msfGFP and rpoB mRNA instead. Assuming a first-order degradation kinetic for the mRNA of both 219 genes, the time profile of the normalized mRNA data can be described with Equation 1. 220 All optimized gene expression constructs with the three tested constitutive promoters resulted in a 268 substantial increase in fluorescence compared with their traditional counterparts (Figure 3 A). 269 While the ranking of the promoter strength was maintained with the optimized gene expression 270 cassettes, absolute differences in the level of expression were reduced as a much higher increase was 271 observed for the weaker Syn35 promoter expressing mCherry (  and R0.5, RNase III restriction sites; gmR, gentamycin resistance gene; tetA, tetracycline resistance 296 gene; ori ColE1 and ori RO1600, origins of replication. 297

Characterization of plasmid-based, inducible fluorescent protein expression 298
Controlled gene expression is required, for instance, when the synthesis of a target product harms cell 299 fitness and needs to be decoupled from growth or in genetic circuits (Chen, 2012;Jusiak et al., 2016;300 Terpe, 2006;Voigt, 2006). We, therefore, chose to further evaluate inducible gene expression by 301 placing the two fluorescent reporter proteins under control of the nagR/PnagAa promoter, inducible 302 with low concentrations of the relatively cheap inducer salicylate (Hüsken et al., 2001). In both, the 303 traditional and optimized expression constructs, the bidirectional terminators were left out as the first 304 attempt to reduce the overall size of the cassette. The removal of the bidirectional terminators should 305 bear similar effects in both cassettes as they have the identical genetic context. Aside from the removal 306 of the bidirectional terminators and the use of a different promoter, both expression cassettes contained 307 the equal genetic parts used for the evaluation of the constitutive promoters (Figure 2 B). 308 Under the control of the inducible nagR/PnagAa promoter, the optimized gene expression cassette 309 showed a similar behavior as with the strong constitutive promoters. The specific fluorescence was 310 increased by 4-fold with the optimized gene expression cassette in comparison to their respective 311 traditional counterparts (Figure 3 B). A similar increase was observed for the msfGFP signal under 312 non-induced conditions whereas for the mCherry constructs only a 2-fold increase was observed. The 313 stable increase of the msfGFP signal under both induced and uninduced conditions supports the concept 314 of a post-transcriptional gene expression enhancement. The lower fold ratio in the non-induced 315 conditions observed with the mCherry constructs could be related to analytical inaccuracies of the 316 weaker mCherry signal. Inducible promoters, such as the nagR/PnagAa promoter system used in this 317 work, tend to have a basal expression which could become problematic for the expression of toxic 318 genes or their use in systems that need to be tightly regulated, e. g., genetic circuits. Attempts to achieve 319 non-leaky inducible expression systems have been made, but their number continues to be limited 320 (Horbal and Luzhetskyy, 2016). We propose the use of the optimized gene expression cassette in 321 known non-leaky inducible promoter setups to increase the available expression range in such systems 322 rather than aiming to engineer a novel non-leaky inducible variant. Since the additional parts of the 323 optimized gene expression cassette tend to act on transcribed mRNA only, an optimized gene 324 expression cassette harboring a tight, inducible promoter system could still exhibit the desired non-325 basal expression in the absence of the inducer and, once induced, reach higher expression levels than 326 the standard counterpart. III is not the major endoribonuclease responsible for mRNA turnover in bacteria, the enzyme does 365 contribute to mRNA degradation. (Deutscher, 2006) (Figure 2 D). The traditional expression 388 cassette was obtained by removing the expression enhancing genetic parts while maintaining the 2 nd 389 RBS of BCD1 upstream of the ilvB and the 2 nd RBS of BCD2 upstream of the aldB (Figure 2 D). 390 Employing the optimized gene expression architecture to the acetoin pathway led to an acetoin 391 accumulation of 5.5 ± 0.76 mM, representing a 2.5-fold production increase when compared to the 392 traditional counterpart (Figure 3 C). The increase of acetoin production solely using the optimized gene 393 expression cassette shows that its use can be extended to production contexts. The optimized gene 394 expression cassette could be advantageous for metabolic engineering approaches where high gene 395 expression is required, such as redirecting native metabolites to the desired production pathway or for 396 enzyme production for in vitro applications. if such a phenomenon occurred in the optimized gene expression cassette, qPCR assays were 405 performed to compare the decay rates of the transcripts. For this purpose, cultivations of the strains 406 expressing msfGFP under the control of the SPA75 promoter were treated with rifampicin and nalidixic 407 acid in the early exponential growth phase, and mRNA samples were retrieved over time. The addition 408 of rifampicin inhibits bacterial RNA polymerase, whereas nalidixic acid inhibits a subunit of the DNA 409 gyrase and topoisomerase. As the mRNA abundance data of the mfsGFP were normalized with the 410 data of the housekeeping gene rpoB to cancel out small differences in the template concentration, the 411 determination of the absolute decay rate of the mfsGFP transcript was not possible. Instead, we 412 calculated the difference between the decay rates of the rpoB and the msfGFP mRNA (i.e., − 413 , with being the decay rate). Given that the rpoB mRNA decay should be equal in both strains 414 the delta can be used to reveal a possible difference in the msfGFP decay rates in the optimized and 415 traditional expression architecture. As hypothesized, the data was significantly higher for the optimized 416 construct (1.6-fold), which translates to a reduced decay rate of the msfGFP transcript (Figure 4). 417 418 Figure 4. qPCR based elucidation of mRNA stability of msfGFP transcripts from the plasmid-based 419 traditional (light-colored) and optimized (dark-colored) expression cassettes harboring the constitutive 420 SPA75 promoter. Early exponential cultivations were treated with the antibiotics, nalidixic acid and 421 rifampicin, to halt DNA replication and transcription, respectively. Samples were taken over time, and 422 mRNA levels were assessed through qPCR. mRNA decay rates of transcripts from each expression 423 cassette were retrieved through the difference between the decay rate of the housekeeping gene rpoB 424 and the target msfGFP. Error bars indicate the standard deviation of two biological replicates. 425 Hence, higher mRNA stability is indeed one factor contributing to the observed increase in 426 fluorescence output and acetoin production. 427

Conclusion 428
In this study, the optimized gene expression cassette architecture proposed by Nielsen et al. was 429 evaluated in P. taiwanensis VLB120 for the expression of fluorescent reporter genes and a 2-step 430 acetoin biosynthesis operon. The optimized gene expression cassette was characterized on a plasmid 431 or single genomic integration basis with either constitutive or inducible promoters to cover all 432 commonly used expression approaches in metabolic engineering. In all evaluations, the optimized gene 433 expression cassette outperformed its traditional counterpart. The highest improvement fold was 434 observed once the RNAse III site was removed and evaluated on a single genomic integration basis 435 under the control of the IPTG inducible PTrc promoter. Such a boost allowed a single genomic 436 integration-based expression to achieve expression levels commonly reached with plasmids. Within 437 the constitutive promoter paradigm, the optimized gene expression cassette increased expression levels 438 of the strongest promoter of a promoter library, showing that this tool could be used to extend 439 expression ranges further. The mRNA transcripts retrieved by the optimized gene expression cassette 440 harnessed higher stability than the transcripts from the traditional counterpart, validating that mRNA 441 stability contributed to the observed results. This work demonstrates the applicability of the optimized 442 gene expression cassette as a tool to achieve high gene expression levels through transcription-443 independent approaches that rely on mRNA stability and translation efficiency. 444 5