Implementation of a Lipase A spore display in Paenibacillus polymyxa.

doi:10.21203/rs.3.rs-3316092/v1

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Implementation of a Lipase A spore display in Paenibacillus polymyxa.

https://doi.org/10.21203/rs.3.rs-3316092/v1

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This study demonstrates the use of a spore display in Paenibacillus polymyxa for the first time, specifically incorporating fluorescent spores expressing the green fluorescent protein (GFP) and the functional display of the lipase A (LipA). Spore display allows the presentation of heterologous proteins on the surface of bacterial spores, offering numerous advantages in various biotechnological applications. The successful implementation of spore display in P. polymyxa involved engineering the spore surface to produce GFP fused to an anchoring protein in the crust, resulting in fluorescent spores that could be readily visualized. After this initial proof-of-concept, LipA from P. polymyxa was heterologously expressed and displayed on the spore surface. The lipase activity was measured over a wide temperature range and an increase in activity up to 70°C was observed. The immobilized catalyst was recycled using simple centrifugation seven times without significant loss of activity.

Paenibacillus polymyxa

spore surface display

lipase A

In an ever-evolving global landscape characterized by an escalating demand for sustainable alternatives to fossil fuel-derived products, biotechnological methodologies have emerged as a highly promising avenue to tackle this pressing concern effectively [1]. With enzymatic catalysis, a wide range of chemicals can be produced efficiently and environmentally friendly [1–3]. Free enzymes normally can be only used once. Therefore, it can be desirable to use methods where the enzymes are immobilized [4–6]. The display of enzymes on the surface of a host organism, such as bacteriophages, bacteria, or yeast cells, has emerged as a powerful tool in protein engineering [7–11]. This technique offers a range of applications in biocatalysis, drug discovery, and bioremediation, allowing for the identification and optimization of enzymatic activities [12, 13]. In the context of enzyme engineering strategies like directed evolution or rational design, enzyme display provides additional advantages due to a phenotype-genotype coupling [14]. It enables the rapid screening of large enzyme variant libraries, offering a high-throughput approach to enzyme optimization and selecting enzymes with specific properties for desired applications [15, 16]. Consequently, enzyme display has become a valuable tool for enhancing the performance and versatility of enzymes, which play crucial roles in various industries and biotechnological fields [5, 17, 18]. Another display technique is autodisplay, which enables the surface expression of a protein of interest on the bacterium Escherichia coli by utilizing the specialized outer membrane protein AIDA-I [19]. This versatile system has applications in protein engineering, drug discovery, and vaccine development [20–22]. Autodisplay in E. coli offers notable advantages compared to other display systems, including user-friendliness, high expression levels, and the capability to display a diverse array of proteins [19]. However, this method has some limitations, such as the requirement for suitable conditions for E. coli growth, which may restrict the experimental setup. Additionally, this technique can’t be used in a pharmaceutical context, whereas the spore surface display is a more robust method that gives a range of usage [23–25]. It has become an exciting approach for showcasing different proteins on the outer layer of bacterial spores, generating considerable interest for its wide range of potential applications in biotechnology [24, 26, 27]. Bacillus subtilis serves as a model organism in terms of sporulation and spore display, and many studies have demonstrated the efficacy of spore display in the expression of various proteins, such as enzymes, antibodies and antigens [27–29]. Enzymes such as lipase, xylanase, and cellulase have been successfully presented on the spore surface, highlighting the potential of spore display for biocatalytic applications [30–32]. While the effectiveness of spore surface display has been successfully demonstrated in Bacillus subtilis, its application in P. polymyxa is still in the early stages [33]. P. polymyxa is a Gram-positive, endospore-forming soil bacterium used as a biofertilizer due to its ability to fix nitrogen and in various biotechnological applications based on its diverse metabolic capabilities and potential for metabolic engineering [34]. Furthermore, P. polymyxa is known for its production of a variety of extracellular enzymes, antibiotics, exopolysaccharides (EPS), and high-value chemicals like R,R-2,3-butanediol in high enantiomeric purity, making it a promising candidate for use in agriculture, industrial biotechnology, and food industry. EPS are synthesized as a barrier against stress factors but also play a role in spore formation [35–38]. Developing spore display in P. polymyxa involves constructing a specific plasmid that can be transformed into the bacterium, enabling the expression and attachment of target proteins onto the spore surface. This is achieved by fusing the desired protein with a spore coat protein which are highly aboundand in the spore coat, and incorporating a flexible linker between the two components [12, 23–27]. Spores can withstand extreme conditions such as high temperatures, radiation, and desiccation, making them highly durable [44]. An additional benefit of spore surface display is the easy recycling of the spores themselves. Compared to conventional methods, this offers several advantages, such as lower energy consumption, lower costs, and improved sustainability, since the spores can be reused over a long period [45].

As proof-of-function in this study, we used GFP as an easily measurable protein to establish the molecular components and continued with a proof-of-concept by displaying a lipase as an industrially relevant class of enzymes that catalyze the hydrolysis of ester bonds in lipids, allowing fats and oils to be broken down into smaller components [46, 47]. They play a crucial role in a wide range of biological processes, from the digestion of dietary lipids to the biosynthesis of complex lipids in living organisms [46]. Additionally, lipases have been utilized in various industrial applications, such as food processing, detergent formulation, and biodiesel production, due to their ability to perform efficient and selective reactions under mild conditions [48]. Applying lipases to the spore surface and using them as immobilized enzymes in an in vitro bioprocess combines the benefits of lipases with spore display, creating a powerful tool for biotechnology [31]. Here, we selected a lipase A (lipoyl synthase EC 2.8.1.8, further called LipA) we identified in the genome of P. polymyxa.

Construction of strain and plasmids expressing the CotE-GFP and CotE-LipA fusion protein on the P. polymyxa spore surface

The spore surface display was designed based on the previously generated knowledge of B. subtilis spore displays [24,26,30]. The display consists of an anchoring protein located in the coat of the spore, a linker for flexibility and distance, and the protein to display. CotE is the corresponding anchoring protein and plays a significant role in assembling the outermost crust layer of B. subtilis. By using the databases Subtiwiki [50] we identified further anchoring proteins known from B. subtilis that are in the crust and performed a blastX-search using the B. subtilis protein sequences against the genome of P. polymyxa DSM 365 [51–53]. By that approach it was observed that cotE is the only cot-gene known in P. polymyxa. [54] The cotE gives a 9.47e^-38 E-value and an identical pairwise coverage of 60.6 % compared to the gene of B. subtilis. The Green Fluorescence Protein (GFP) was chosen for the proof-of-function as it gives a readily measurable output. A His-Tag was added to the N-terminus of the fusion protein, if future purification will be necessary. The construct was designed with the native promotor of cotE, P_cotE, to synchronize the spore formation with the expression of the fusion protein and, hence, the incorporation into the spore crust layer. For the native promotor 500 bp upstream the cotE gene in which a RBS was found, were selected as the putative promotor region. The fact that the CotE is translated in an early stage of sporulation, the composition of the spore crust layers could be disordered or may lead to an incomplete or changed composition. As a plasmid backbone, the pHeip shuttle vector was used as it has a medium-high copy number known to function in P. polymyxa [55,56]. Besides the CotE-GFP spore display we decided to create a spore display with a lipase as A displayed enzyme. In literature spore display was already performed in B. subtilis with its lipase A [31]. We searched analogous to the search for the cotE anchoring protein in the P. polymyxa genome for a similar gene using the LipA-sequence from B. subtilis as our query. A Lipoyl synthase with a 2.83e^-128 E-value and an identical pairwise coverage of 63.7 % in the P. polymyxa genome. For simplicity we annotated it as Lipase A. Additionally, we compared with TM align and ColabFold the structural similarity’s [57,58]. This revealed a TM-score of around 0.94 which strongly indicates that the structure of the lipases of B. subtilis and P. polymyxa are about the same fold and (see Supplement Figure 1). Accordingly, we chose assay parameters as described for LipA from B. subtilis [32]. Sporulation is frequently induced via media change or the usage of sporulation media like SG-Media [30,31]. For our studies, we used minimal media based on the M9 media with additives described in the material and methods part giving us a faster sporulation without formation of cell-clumbs as frequently occurred with SG-media (data not shown).

Verification of the GFP display via Fluorescence microscopy and flow cytometry

The proof-of-function is shown in Figure 1. The merged image shows green fluorescent purified spores. Washing steps would have resulted in removing unbound GFP, reducing signal intensity. The result indicates that the chosen expression system and the fusion protein are correctly expressed during sporulation and that the incorporation into the crust was successful. Cytometric analysis was performed to cross-check the image, as shown in Figure 2.

Plots A – C in figure 2 show microscope images of wild type cells (A), wild type spores (B) and fluorescent spores (C) carrying the CotE-GFP. Plots D – F show plots where the fluorescence signal (FITC-H channel) is plotted against the counted number. The cytometric analysis of the wild type cells (D) and spores (E) as control shows no fluorescence signal in the FITC-H channel compared to F. In F, the fluorescence signal verifies the microscopic images and, therefore, the presence of GFP on the spore surface.

The result indicates that the expression system is active and verified via fluorescence microscopy and flow cytometry.

For further investigations on the spore display we incorporated a TEV-cleavage site (ENLYFQ ▼(S/G/A/M/C/H) between the linker and the protein of interest, here, GFP. The results in Figure 3 show a difference in the untreated and treated supernatant, showing that the TEV-cleavage site is accessible for the protease. Consequently, we showed that at least a part of the GFP is covalent bound to the spore. This result is additionally confirmed via SDS-page (Supplement Figure 4).

Catalytic performance of the CotE-LipA fusion protein

After validating the expression of GFP via the spore display, the next step was the expression of functional enzymes displayed on the surface of P. polymyxa spores. Hydrolytic activity of purified spores displaying a C- and N-terminal fusion protein of lipase A is shown in Figure 4. The samples have been incubated at different temperatures. The first experiment was based on the lipase assay described by Hui et al. 2022 [32] and showed hydrolysis activity for both constructs. The activities vary concerning the respective substrate. For the long chained fatty acid, 4-Nitrophenyl-palmitate and 4-Nitrophenyl-dodecanoic, the absorbance levels were at the level of the background and the substrate control (Supplement Figure 1). For the short chained fatty acid 4-Nitrophenyl-octanoate the hydrolysis activities were significantly higher compared to the background, the substrate control and to longer chain fatty acids. After this first promising results at 42°C, and the knowledge that spore display can enhance enzyme stability [43] this was also tested it with our system. Both C- and N-terminal displayed LipA show higher activities with increasing temperatures. We observed significant changes in the absorbance at 410 nm for every measurement in Figure 4 with the C8 substrate compared to the wild type spores. Besides the temperature experiments, the effect of the duration of the experiment was investigated. Therefore, a 16 h experiment was performed. This experiment showed an increase in the absorbance levels for both constructs without an increase in the respective controls (Supplement Figure 2). As previous literature shows, LipA is more active for substrates with a medium chain length of fatty acids.[59] The activity for the wild type spores not displaying LipA can be explained by the autohydrolysis of the substrate with increasing temperatures as shown in Figure 5. The absorbance levels in Figure 4 let us assume that the spore surface display of P. polymyxa has a high thermostability.

Therefore, the temperature was raised to 70 °C, and the specific absorbance of the cleaved substrate is given in Figure 5. Only the C8 substrate (4-Nitrophenyl-octanoate) was used for the temperature optimization because it showed the highest absorbance levels with the CotE-LipA spore display. The fact that the substrate control and the wild type spores have nearly the same low absorbance levels indicates that the CotE-LipA C- and N-terminal have higher activities at higher temperatures.

A direct comparison of C- and N-terminal fusion proteins showed a slightly different absorbance and less hydrolytic activity with the C8 substrate while as shown in Figure 4 no significant difference of activity could be observed for the two fusions with the C12 fatty acid.

Kinetic calculations for CotE-LipA hydrolysis of C8 substrate

In addition, a Michael-Menten kinetic study was performed to investigate the catalytic performance of the CotE-LipA fusion protein. Kinetics of only C8-substrate were analyzed due to low the low activities with the C12 and C16. The results are shown in Figure 6.

The V_max results in a maximum velocity of 0.3 nmol mL^-1 min^-1 and the K_m has a value of 2.6 mM. A comparison of these results to free LipA enzyme was out of scope of this study since P. polymyxa produces several lipases and esterase’s, which made a direct comparison not possible.

Recyclability of spores and reuse

As the ease of spore purification is a major advantage of spore display, we wanted to assess the potential recyclability of our system, like the recyclability demonstrated in the B. subtilis spore display. Quantitative analysis revealed that the recycled spores maintained their function as an immobilization particle after seven cycles (Figure 7). The activity of the immobilized enzymes remained unaffected, indicating no degradation of the displayed enzyme on the spore surface and the retention of their binding capacity for the target enzyme. Exemplary the recycling of the C- and N-terminal CotE-LipA spores is shown over seven runs with the substrate 4-Nitrophenyl-octanoate.

We have successfully developed a spore surface display system for P. polymyxa DSM 365, utilizing an analog to the version of the spore surface display technology employed in B. subtilis [32]. Our findings demonstrate the system’s functionality through the fluorescence signal of CotE-GFP spores and catalytic activity of LipA when fused to the anchoring protein. In a recently published manuscript about spore surface display in B. subtilis, the researchers observed a 50% activity loss over 50°C with the LipA displayed [31]. Yet, we observed increasing activity with rising temperatures which can be hypothesized to either a more robust spore surface display or because the lipase A of P. polymyxa is more thermostable or a combination of the two. Furthermore, we observed a twice as high hydrolytic activity with short-chain fatty acids C8 in the assay for lipase A compared with long-chain fatty acids (C12, C16). The kinetic measurements with immobilized enzyme cannot readily be compared with the kinetics from free enzymes. The influence of the activity of the immobilized enzyme can only be assumed. The recyclability of the immobilized enzymes further highlights their stability and potential as robust biocatalyst [61]. We observed no decrease in hydrolytic activity over seven runs. Our study serves as a foundation for expanding the application of spore surface display in P. polymyxa. Investigations of different linkers and promoters could further enhance the activity of displayed proteins. Additionally, the spore surface display was used for the first time with a TEV-cleavage site between the linker and the protein. This enabled us to prove that the displayed enzymes are not just absorbed by the spores but are a real fusion protein incorporated into the spore crust. This cleavage site facilitates the surface expression of diverse proteins on spores, enabling their facile purification. However, the system’s current cleavage efficiency is not optimal and requires further enhancements to achieve maximum efficacy. Future advancements could allow the simultaneous expression of multiple enzyme types, facilitating the engineering of enzymatic cascades on a single spore surface and creating versatile biocatalysts capable of multiple chemical transformations like it’s already described for B. subtilis [30,62]. These developments are expected to contribute significantly to the field of sustainable catalysis.

In this study, we have demonstrated the suitability of the P. polymyxa spores for immobilization of enzymes. Our spore display approach offers several advantages, including cost, self-immobilization, high enzyme stability, and the ability to easily recover the immobilized enzymes for multiple cycles of use. Immobilization also improves protein handling and allows rapid separation of immobilized enzymes from the reaction product. In addition, spore display by P. polymyxa offers economic advantages, longer shelf life, and resistance to solvents compared to free enzymes. As demonstrated in our study, this spore display approach provides significant advantages for protein immobilization and reuse. It could be used for testing different protein-displaying spores in a standardized manner. For further characterization of the lipase A of P. polymyxa, the free enzyme needs to be compared to the spore display. Furthermore, other substrates need to be tested to demonstrate real application outside the lab. Overall, our results highlight the potential of spore display as a versatile tool for protein immobilization and offer a promising avenue for further research and application development in this field.

Bacterial Strain and Growth Conditions

P. polymyxa DSM 365 was obtained from the German Collection of Microorganisms and Cell Culture (DSMZ), Germany. Plasmid cloning and multiplication were performed in Escherichia coli Turbo (New England Biolabs, USA). E. coli S17-1 (ATCC 47055) was used as a conjugative donor strain to mediate the transformation of P. polymyxa. The strains were cultivated in LB media (10 g/L peptone, 5 g/L yeast extract, and 5 g/L NaCl). For plate media, an additional 1.5 % of agar was used. The media were supplemented with 50 μg/mL neomycin and 20 μg/L polymyxin if required. P. polymyxa was cultivated at 30°C, while E. coli was cultivated at 37°C unless stated otherwise. For liquid culture, the strains were cultivated in 3 mL of LB media in 13 mL culture tubes and incubated at 250 rpm. The strains were stored as cryo-cultures in 24 % glycerol and kept at −80°C for more extended storage.

Strain and Plasmid Construction

The plasmids used in this study use the same backbone of the pHeip vector [63]. The Backbone with the respective overhangs to the construct was amplified via PCR by use of Q5-polymerase with the pHeip vector. The anchoring protein CotE and the LipA gene were amplified via Q5-PCR from genomic DNA of P. polymyxa DSM 365. The target proteins were fused C- and N-terminal to the anchor protein via a flexible Glycine/Serine (GGGGGSGGGGG) peptide linker. In most cases, the anchoring and fusion protein’s linker is necessary for displaying enzymes and keeping their activity. [2, 3]. The constructed plasmids contain a resistance gene against neomycin and an origin of replication for E. coli and P. polymyxa. Genbank files of the plasmids and strain lists are provided in the supplementary information.

Sporulation and Purification

Sporulation of both wild type and mutant P. polymyxa strains was induced using nutrient-deficient media, M9. The M9 consists of M9 5 x salts (KH₂PO₄ 3 g/L, NaCl 0.5 g/L, Na₂HPO₄ 6.78 g/L, NH₄Cl 1 g/L), glucose monohydrate 4 g/L, MgSO₄ 1mM, CaCl₂ 0.1M, Vitamin solution 2 mL/L and Trace elements solution 1 mL/L (composition in supplementary information). Each starter culture was inoculated with a single colony in 50 mL M9 medium in a 250 ml Erlenmeyer flask and incubated overnight at 37°C. The main culture was then inoculated when the optical density at 600 nm (OD₆₀₀) reached 0.1. The incubation time varied for different variants based on their respective growth rates and was individually optimized. The cells were harvested by centrifugation at 3000 x g for 30 min, and the resulting cell pellets were resuspended in an equal volume of phosphate-buffered saline (1 x PBS, pH 7.4 composition in supplementary information) treated with 50 μg/mL lysozyme for 1 h at 37°C. Finally, the harvested spores were washed twice with PBS, and their morphology was examined using a fluorescence microscope.

Fluorescence Microscopy and Image Analysis

A 3 μL aliquot of a CotE-GFP spore suspension in PBS was dropped onto a microscope slide, covered with a coverslip, and mounted on a Zeiss Imager Fluorescent microscope. A 100× phase contrast objective and a 488 nm laser line (100 ms exposure time, 3 % laser) were used to image the spores. Images were exported and processed using ImageJ (NIH)[65].

Cytometric analysis

To verify the fluorescent spores and to quantify the respective number of spores per OD the cytometer (BD Plus6) was used. 195 μL purified spore solution was mixed with 50 μL Beads (Rainbow calibration 8peaks Beads, BD Bioscience) with a defined concentration of 10⁷/mL and were measured in triplicates via the cytometer. A threshold of 75,000 in the FCS scatter was set. The acquisition was stopped when either the bead number in gate R1 (respectively for the normalization and counting of the spores) counted 10,000 or after 2 min. This method was adapted from [66].

Gates, indicated in red in Supplement Figure 3, were set to distinguish between cells, spores, and debris while analyzing the spores with the flow cytometer. Gate R4 for debris and P1 for cells was defined using scatter plot A, which shows a sample of a fresh overnight culture of P. polymyxa (Supplement Figure 3). Plot B shows only purified wild type spores, so the gate for spores was defined as spore gate P2. In the scatter plots, the forward scatter (FSC-H) is plotted against the side scatter (SSC-H) to see the population differences. After setting the gates, they will be fixed for every follow-up measurement to ensure comparability.

Lipase activity assay

Spores with CotE-LipA were harvested as described above and resuspended in Tris-HCL buffer (50 mM pH 7.5). Lipase activity was determined using a slightly adapted pNPP-method previously established by [49]. Each substrate C8, C12, and C16 was prepared as a 10 mM stock solution in isopropanol. The OD₆₀₀ of the spores in the reaction volume was set to 1 using a cuvette with 1 cm path length, and 1mM substrate was added to a final volume of 1 mL the standard reaction was carried out for 1 h at 42°C and 800 rpm agitation. Besides these, reactions with higher temperatures were carried out with the same conditions as the desired temperature. The reaction mix was spun down after 1 h, and 100 µL of the supernatant was loaded in triplicates on a 96-well microtiter plate (GREINER, flat, transparent Item. No.: 655101). The absorbance of the corresponding product was measured at 410 nm with a microplate reader (TECAN^®).

TEV-cleavage assay

Cleavage activity was evaluated with spores of CotE-GFP with the TEV-cleavage site between the linker and GFP. The spores were diluted to a final OD₆₀₀ of 1 using a cuvette with 1 cm path length, and the assay was adapted and performed according to the manufacturer’s instructions (New England BioLabs^®). After the incubation of 16 h at 30°C, a 10 µL sample was taken and 1 to 10 diluted into 96-well black microtiter plates (GREINER flat, black Item No.: 655076). After this, the tubes were spun down, and a 10 µl sample was taken and 1 to 10 diluted in the same plate. Fluorescence was measured with a microplate reader (TECAN^®) excitation 485 nm and emission 510 nm with a gain of 115.

Recycling of spore activity

For the repeated, continuous usage of the spores, simple centrifugation (10 min, 20.000 x g) of the used recombinant (CotE-LipA) spore, washing with PBS buffer, and resuspension into a newly prepared substrate containing reaction buffer was performed.

Statistical analysis

All experiment data were plotted with GraphPad PRISM with the ± standard error of the mean. All assays were performed for at least three replicates. The kinetics assay was fitted to the Michaelis-Menten model. Statistical significance and P values were derived from two-tailed t-tests.

Ethics declarations

Not applicable.

Consent for publication

Not applicable.

Availability of Data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study is part of the German Federal Ministry of Education and Research (BMBF) funded project Polymore with the no. 031B0855A.

Authors’ contributions

MZ, JS and JK conceived and designed the experiments; MZ carried out all of the experimental work; MZ wrote the manuscript. MZ, JS, JK revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Implementation of a Lipase A spore display in Paenibacillus polymyxa.

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