Implementation of spore display in Paenibacillus polymyxa with different hydrolytic enzymes

doi:10.21203/rs.3.rs-3785947/v1

Download PDF

Research Article

Implementation of spore display in Paenibacillus polymyxa with different hydrolytic enzymes

https://doi.org/10.21203/rs.3.rs-3785947/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa,achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase are expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV-cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range.

Paenibacillus polymyxa

spore surface display

lipase

esterase

Biotechnological methods have emerged as promising ways to effectively address pressing issues like the energy crisis and global warming in an ever-evolving global landscape characterized by increasing waste production and demand for sustainably produced chemicals [1]. Purely enzymatic process variants can outperform classical processes by maximizing substrate conversion rates and yields by accounting for losses to maintain cell metabolism. Via enzymatic catalysis, a wide range of substances can be produced efficiently under environmentally friendly conditions, such as low temperatures and atmospheric pressure, as well as in aqueous solutions [1–3]. However, these methods also have limitations. For example, free enzymatic biocatalysts can usually only be used once, but process variants in which the enzymes are immobilized can solve this problem [4–6].

The display of enzymes on the surface of a host organism, such as bacteriophages, bacteria, yeast cells, or the spore display, has emerged as a powerful tool in protein immobilization [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 genotype-phenotype 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]. One such 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. An alternative method is the spore surface display, in which the endospores of spore-producing bacteria are used as carriers for the immobilized proteins. Spores can withstand extreme conditions such as high temperatures, radiation, and desiccation, making them highly durable [23]. B. 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 [24–26]. Enzymes such as lipases, xylanases, and cellulases have been successfully presented on the spore surface, highlighting the potential of spore display for biocatalytic applications [24, 27, 28]. The high robustness and the genotype-phenotype preservation under harsh conditions of this technique give major advantages [29–31]. An additional benefit of spore surface display is the easy recycling of the spores themselves by simple separation through e.g. centrifugation and the reuse of the spores. 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 [32]. It has become a promising 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, 30, 33]. While the efficacy of spore surface display has been successfully demonstrated in B. subtilis, its general application to P. polymyxa has recently been patented, but there are no publications on its application yet. This publication marks a starting point [34].

P. polymyxa is a Gram-positive, endospore-forming soil bacterium which is used as a biofertilizer commercially due to its ability to fix nitrogen. In addition, it has a high potential for various biotechnological applications due to its diverse metabolic capabilities and currently optimized genetic accessibility in combination with a Generally Recognized As Safe (GRAS) status for several applications [35]. By that, P. polymyxa is known for its production of a variety of extracellular enzymes, antibiotics, exopolysaccharides (EPS), and high-value-added chemicals like R,R-2,3butanediol, which renders it a promising candidate for utilization in agriculture, white biotechnology, and food industry. However, the high production yields required for an industrial process lead to the problem of product toxicity. Developing spore display in P. polymyxa would add a not yet-described tool to the scientific community to its repertoire of advantages and tackle the toxicity problems. To achieve a working spore display in P. polymyxa, we constructed a specific plasmid to enable the expression and attachment of target proteins onto the spore surface. This was accomplished by fusing the desired protein to a spore coat protein and incorporating a flexible linker between the two components [18, 36–40]. To verify that the constructed system worked, we used GFP as a reporter system to establish the basic molecular components as proof-of-principle in this study. As a proof-of-concept, we presented a native and a heterologous lipase, as well as an esterase and a lipoyl synthase on the surface of the spore. These enzymes belong to an industrially important class that catalyzes the hydrolysis of ester bonds in lipids, facilitating the breakdown of fats and oils into smaller components [41, 42]. 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 [43]. This study thus lays the foundation for P. polymyxa spore display technology and can contribute to the way we harness the natural repertoire of the bacterium for a sustainable biotechnological future, with displayed enzymes paving the way for innovative and environmentally friendly industrial processes.

Construction of strains and plasmids for the expression of fusion proteins on the spore surface

The spore surface display was designed based on the previous knowledge obtained from B. subtilis spore display approaches [24, 30, 33]. The display system includes an anchoring protein located in the coat of the spore, a linker for flexibility and distance, and the displayed protein. By using the database SubtiWiki [45] we identified anchoring proteins known from B. subtilis and performed a blastX-search using the B. subtilis protein sequences against the genome of P. polymyxa DSM 365 [46–48]. By that approach, we could identify cotE as the only cot-gene identified in P. polymyxa [49]. CotE plays a significant role in assembling the outermost spore layer, called the crust, of B. subtilis and the homolog protein from P. polymyxa showed an E-value of 9.47 e^− 38 and a query coverage of 71.27%.

First, GFP was chosen for the proof-of-principle as it gives a readily measurable output. A His-Tag was added to the C-terminus of the fusion protein for future purification purposes. The medium-high copy number pHEiP shuttle vector, already developed for P. polymyxa, was chosen as a backbone [50, 51]. The construct was designed with the native promoter of cotE from P. polymyxa, P_cotE, to synchronize the spore formation with the expression of the fusion protein and, hence, the incorporation into the spore’s crust layer. For the native promoter 500 bp upstream the cotE gene were selected as the putative promoter region.

In addition to the CotE-GFP spore display, we created a spore display with a native lipase, an esterase from P. polymyxa, and a heterologous lipase from B. subtilis. In literature, spore display was already performed in B. subtilis with its native lipase A [27]. We blasted for lipases known from B. subtilis in the P. polymyxa genome. No analog to lipase A (P37957) was found in the P. polymyxa genome. We thus used the lipA-sequence from B. subtilis and used it for the spore display as a heterologous gene. The bioinformatic search provided us with a monoglyceride lipase (Lip3, NF024107.3) as well as a para-nitrobenzyl esterase (PnbA, P37967) which were chosen for the display of two native enzymes from P. polymyxa. Assay parameters were chosen as described for LipA from B. subtilis [28]. P. polymyxa strains were transformed with corresponding plasmids verified through DNA sequencing for spore production.

Identification of the GFP display via fluorescence microscopy and flow cytometry

The proof-of-function is shown in Fig. 1. The merged image of purified and washed spores shows a signal in the channel corresponding to green fluorescence. The results indicate that the chosen expression system and the fusion protein are correctly expressed during sporulation and that GFP is bound to the spore surface. Cytometric analysis was performed to verify fluorescence on the spores, as shown in Fig. 2.

Microscopic images of wild type vegetative cells (A) wild type spores (B) and fluorescent spores (C) carrying the CotE-GFP are shown in Fig. 2. Plots D – F show the fluorescence signal (FITC-H channel) plotted against the event count. The cytometric analysis of the wild type cells (D) and spores (E) as controls shows no fluorescence signal in the FITC-H channel compared to F, where GFP displaying spores were analyzed.

These results indicate that the expression system is active as verified via fluorescence microscopy and flow cytometry.

For further investigations of the spore display we incorporated a TEV-cleavage site [52] between the linker and the protein of interest, here, GFP. The results in Fig. 3 show a difference between the untreated and treated supernatant, indicating that the TEV-cleavage site is accessible for the protease. Consequently, we showed that at least a part of the GFP is covalently bound to the spore. This result was additionally confirmed via SDS-PAGE (Supplement Fig. 5).

Enzyme activity assays of the CotE-Lip3, CotE-LipA, and CotE-PnbA fusion proteins

After validating GFP expression via spore display, the next step was to express industrially relevant enzymes on the surface of P. polymyxa spores. The hydrolytic activity at different temperatures of purified spores expressing either lipase or esterase is shown in Fig. 4. The first experiment was based on the lipase assay described by Hui et al. 2022 [28], conducted with an OD₆₀₀ of 1 of spores, and showed hydrolysis activity for all constructs at 42°C.

The absorbance at 410 nm is directly correlated to the activity of the spore display based on the standard curve of p-nitrophenol (Supplement Fig. 3). The activities for all three displays varied concerning the respective substrate. For the long-chained fatty acid, 4-Nitrophenyl-palmitate (C16) and 4-Nitrophenyl-dodecanoic (C12), the absorbances were at the level of the background and the substrate control (Supplement Fig. 1 and Supplement Fig. 2). For the short-chained fatty acid 4-Nitrophenyl-octanoate (C8), the hydrolysis activities were significantly higher compared to the background, the substrate control, and longer chain fatty acids. After these first promising results at 42°C, we tested our system with varying temperatures in a range from 4°C to 70°C.

The activity of the displayed esterase PnbA reached its optimum at 4°C. The activity was 6-fold higher compared to the wild type spores. Correlating the absorbance levels with the activity, the PnbA reaches 158 U [µm of product/hour]. The LipA from B. subtilis showed the highest activity with 116 U [µm of product/hour] at 42°C 4.4-fold higher activity compared to the wild type spores. The displayed Lip3 showed higher hydrolysis activity with rising temperature. It reached 29 U [µm of product/hour] which is 2.4-fold higher activity compared to the wild type spores at 70°C. Figure 4 shows the absorbance levels of all three displayed enzymes with the C8 substrate compared to the wild type spores and the substrate control. The activity for the wild type spores not displaying any enzyme can be explained by the autohydrolysis of the substrate with increasing temperatures, as shown in Fig. 4. Figure 4 shows that, with the exception of Lip3, the assayed enzymes' activity decreased from 42°C to 50°C. In order to evaluate higher temperature ranges for Lip3, the assay temperature was increased to 70°C. Only the C8 substrate was used for the high-temperature assay because it showed the highest absorbance levels with all three enzymes on the spore display (Supplement Fig. 2). The Lip3 doubled its activity from 40°C to 70°C.

The activity of the LipA dropped from 42°C to 60°C to half of the maximum activity but kept this activity also at 70°C and reached absorbance levels like the wild type spores.

Recyclability and reusability of the spore display

As the ease of spore purification is a major advantage of spore display, we wanted to assess the potential recyclability of our system, as demonstrated also in the B. subtilis spore display with simple centrifugation and washing steps [adapted from 28]. Exemplary recycling of CotE-LipA, CotE-Lip3, and CotE-PnbA spores is shown over 10 runs with the substrate 4-Nitrophenyl-octanoate. The experiment was performed as described for the lipase assay. The spore display for the Lip3 maintained its low activity as an immobilization particle after 10 cycles (Supplement Fig. 3). The activity of the immobilized enzymes, PnbA and LipA, dropped to a low activity. The CotE-LipA construct lost 45% hydrolytic activity after the first run and an additional 26% from runs 2 to 3. The CotE-PnbA lost 18% after the first and an additional 19% from run 2 to 3. After the third run the activities of all three enzymes stayed the same for 4 runs dropping then nearly to the background activity from the wild type spores and the substrate control (Supplement Fig. 3, Table 1).

Table 1

Recycling runs of all three spore displayed enzymes with their respective loss in activity after each run.
Recycling Runs	Lip3 [A410 nm]	Change in %	PnbA [A410 nm]	Change in %	LipA [A410 nm]	Change in %
1	3,4433E-01	/	6,8187E-01	/	1,1549E + 00	/
2	3,9179E-01	-14%	5,6180E-01	18%	6,3360E-01	45%
3	3,8470E-01	2%	4,5480E-01	19%	4,7157E-01	26%
4	3,5587E-01	7%	4,4443E-01	2%	4,6197E-01	2%
5	3,4897E-01	2%	4,2273E-01	5%	4,2637E-01	8%
6	3,4707E-01	1%	4,0180E-01	5%	4,1923E-01	2%
7	3,1297E-01	10%	3,6830E-01	8%	3,9290E-01	6%
8	2,8870E-01	8%	3,4555E-01	6%	3,3160E-01	16%
9	2,7643E-01	4%	2,9600E-01	14%	2,6603E-01	20%
10	2,6287E-01	5%	2,5137E-01	15%	2,4897E-01	6%

Unexpected hydrolytic side activity of the Lipoyl synthase A displayed on P. polymyxa spores

The lipoyl synthase A, which belongs in contrast to the lipases and esterases to the class of synthases/transferases is abbreviated as lipA in the well-structured SubtiWiki [45], which is the abbreviation used for lipase A from B. subtilis in other major databases such as UniProt where both lipase A and lipoyl synthase A are abbreviated as lipA. This inconsistent nomenclature made us assay this protein as a lipase, until the mistake was recognized. However, as an unexpected result, this enzyme showed hydrolytic activity for the p-nitrophenyl caprylate (pNPC, C8) substrate used for the lipase assays. Furthermore, the lipoyl synthase showed increased activity in this assay with increasing temperature. This activity is likely not to be accentuated by autohydrolysis or the spores themselves, as can be seen in Fig. 5. The lipoyl synthase reached a maximum absorbance level at 410 nm of 1.04 which is 4.99-fold higher compared to the control. The correlation to the standard curve of p-nitrophenol gives 101 U [µm of product/hour].

Additionally, we tested the recycling abilities of the lipoyl synthase and observed no significant loss in hydrolysis side activity over seven runs.

We have successfully developed a spore surface display system for P. polymyxa DSM 365, utilizing an analog version of the spore surface display technology employed in B. subtilis [28]. Our findings demonstrate the system’s functionality through the fluorescence signal of CotE-GFP spores, the catalytic activity of two different lipases, and an esterase 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 [27]. With rising temperatures, we saw a loss in activity for the lipases and esterases but an increase in the measured activity of the lipoyl synthase. The lipoyl synthase has so far not been described as an iron-sulfur cluster enzyme required for the synthesis of lipoic acid, during which two sulfur are transferred to an octanoic acid [53]. As both the substrate for the synthesis and the hydrolysis contains a C8-fatty acid, making a binding in the enzyme pocket likely, the observed hydrolytic could represent an example of substrate promiscuity [54], yet further experiments are required to confirm this. Previous studies showed that lipases and esterases used preferably small- to medium-chain fatty acids. We can confirm the same feature with the enzymes displayed in our system [55]. In addition, we observed for the first time a high hydrolytic activity at 4°C for the esterase PnbA from P. polymyxa. Cold-active esterases are described in different bacterial species but not from P. polymyxa [44, 54, 55]. P polymyxa is known to have a cold-active pullulanase [58]. Future work should use the free enzymes to determine which property changes arise from the immobilized forms. Concerning the recyclability properties of all four spore displays, the LipA shows a drop-in activity after the first run to its half. Meanwhile, Lip3 activity remained in the same low range during recycling. The PnbA loses 18% of its activity after the first and an additional 19% after the second. In contrast to the two native and one heterology-expressed enzymes, the recyclability of the lipoyl synthase from P. polymyxa showed no significant loss in activity over seven runs (Fig. 6). Our study serves as a foundation for expanding the application of spore surface display to P. polymyxa. Investigations of different linkers and promoters could further enhance the activity of displayed proteins. A previous study achieved an increase in the protein load on the spore surface and the activity of this system by knocking out extracellular proteases in B. subtilis, which would also be beneficial in P. polymyxa [28].

Additionally, in this study 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 show that the displayed enzymes are not just absorbed by the spores, but are a real fusion protein incorporated into the spore crust. This approach could enable the use of surface expression of diverse proteins on spores as a simple protein purification method. However, the system’s current cleavage efficiency is not optimal and requires further enhancements to achieve maximum efficacy. Future studies 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 [24, 60]. These developments are expected to contribute significantly to the field of sustainable biocatalysis.

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, 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 offers economic advantages, longer shelf life, and resistance to solvents compared to free enzymes [61]. 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. The free enzymes need to be compared to the spore display for further characterization of the lipases and the esterase of P. polymyxa. Additionally, the lipoyl synthase should be assayed for synthase activity while immobilized, to examine the effects of spore display on this enzyme. Overall, our results highlight the potential of P. polymyxa 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 E. 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 [62]. The Backbone with the respective overhangs to the construct was amplified via PCR by use of Q5-polymerase (New England Biolabs, USA) with the pHEiP vector. The anchoring protein CotE and the lipase genes were amplified via Q5-PCR from genomic DNA of P. polymyxa DSM 365. The target proteins were fused 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 is frequently induced via media change or the usage of sporulation media like SG-Media [24, 27]. 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-clumps as frequently occurred with SG-media (data not shown). 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. For rapid small-scale sporulation, agar plates could be used as follows. 200 µl of a preculture grown in LB were plated on M9 minimal media (see above) agar (1.5% agar) and incubated at 30°C for at least two days until a biofilm was observed. The amount of sporulated cells was checked by light microscopy. The spore containing biofilm was removed with a spatula, resuspended in PBS and washed as described above.

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) [64].

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 [65].

Gates, indicated in red in Supplement Fig. 4, 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 Fig. 4). 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.

Hydrolysis activity assay

Spores with the fusion protein 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 [44]. 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 a 1 cm path length, and 1 mM 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. The reaction mix was spun down (10 min, 20.000 x g at 20°C) 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, USA After the incubation of 16 h at 30°C, a 10 µL sample was taken and 1 to 10 diluted into 96well black microtiter plates (GREINER flat, black Item No.: 655076). After this, the tubes were spun down (10 min, 20.000 x g at 20°C), and a 10 µl sample was taken and 1 to 10 diluted in the same plate. Fluorescence was measured with a microplate reader (TECAN®) with an excitation of 485 nm and an emission of 510 nm with a gain of 115.

SDS-Polyacrylamide-Gel electrophorese (SDS-PAGE)

SDS-PAGE was used to separate proteins by molecular weight to analyze the molecular weight and purity of the recombinant proteins. Discontinuous SDS gels containing a 4% recovery gel and a 10% separation gel were used for better separation. First, the separating gel (approximately 3.5 mL) was poured and covered with 1 mL of water. After the polymerization of the separating gel, the water was poured off, and the collecting gel was poured on top of the separating gel. The sample comb was then placed in the gel. The SDS gel was loaded into the Mini-Protean Tetra Cell (Bio-Rad) and covered with running buffer. 5–10 µL of protein sample was mixed with 6 µL ddH2O and 4 µL 4 x SDS application buffer and incubated at 95°C for 5 minutes. The protein samples and 5 µL of protein size standard were then applied to the SDS gel. The proteins were separated at 20 mV until the blue band containing the dye from the application buffer disappeared from the gel. The gel was in a staining solution for 30 min and then destained twice for 15 min in a destaining solution. To highlight the protein bands, the gel was then destained in H₂O overnight.

Recycling of spore activity

For the repeated, continuous usage of the spores, centrifugation (10 min, 20.000 x g at 20°C) of the used recombinant (CotE-Lip3, CotE-PnbA, CotE-LipA, and CotE-LipoylA) 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. 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

JK would like to dedicate this publication to Uwe Bornscheuer for his 60^th Birthday.

Papadopoulou C-I, Loizou E, Chatzitheodoridis F. Priorities in Bioeconomy Strategies: A Systematic Literature Review. Energies. 2022;15:7258.
Ramos MDN, Milessi TS, Candido RG, Mendes AA, Aguiar A. Enzymatic catalysis as a tool in biofuels production in Brazil: Current status and perspectives. Energy Sustain Dev. 2022;68:103–19.
Peng Y, Chen Z, Xu J, Wu Q. Recent Advances in Photobiocatalysis for Selective Organic Synthesis. Org Process Res Dev. 2022;26:1900–13.
Nunes YL, de Menezes FL, de Sousa IG, Cavalcante ALG, Cavalcante FTT, da Silva Moreira K, et al. Chemical and physical Chitosan modification for designing enzymatic industrial biocatalysts: How to choose the best strategy? Int J Biol Macromol. 2021;181:1124–70.
Bilal M, Asgher M, Cheng H, Yan Y, Iqbal HMN. Multi-point enzyme immobilization, surface chemistry, and novel platforms: a paradigm shift in biocatalyst design. Crit Rev Biotechnol. 2019;39:202–19.
Remonatto D, Miotti Jr. RH, Monti R, Bassan JC, de Paula AV. Applications of immobilized lipases in enzymatic reactors: A review. Process Biochem. 2022;114:1–20.
A. Sheldon R, Basso A, Brady D. New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy. Chem Soc Rev. 2021;50:5850–62.
Chen I, Dorr BM, Liu DR. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci. 2011;108:11399–404.
Hosse RJ, Rothe A, Power BE. A new generation of protein display scaffolds for molecular recognition. Protein Sci. 2006;15:14–27.
Bamba T, Guirimand G, Kondo A, Hasunuma T. Enzyme display technology for lignocellulosic biomass valorization by yeast cell factories. Curr Opin Green Sustain Chem. 2022;33:100584.
Ghaemmaghami S, Huh W-K, Bower K, Howson RW, Belle A, Dephoure N, et al. Global analysis of protein expression in yeast. Nature. 2003;425:737–41.
Sharma A, Gupta G, Ahmad T, Mansoor S, Kaur B. Enzyme Engineering: Current Trends and Future Perspectives. Food Rev Int. 2021;37:121–54.
Chowdhury R, Maranas CD. From directed evolution to computational enzyme engineering—A review. AIChE J. 2020;66:e16847.
Guirimand GGY, Bamba T, Matsuda M, Inokuma K, Morita K, Kitada Y, et al. Combined cell surface display of β-D-glucosidase (BGL), maltose transporter (MAL11) and overexpression of cytosolic xylose reductase (XR) in Saccharomyces cerevisiae enhance cellobiose/xylose co-utilization for xylitol bio-production from lignocellulosic biomass. Biotechnol J. 0 ja:1800704.
Aharoni A, Griffiths AD, Tawfik DS. High-throughput screens and selections of enzyme-encoding genes. Curr Opin Chem Biol. 2005;9:210–6.
Mandeep, Gupta GK, Shukla P. Enzyme Engineering Techniques for Biotechnological Applications. In: Shukla P, editor. Microbial Enzymes and Biotechniques: Interdisciplinary Perspectives. Singapore: Springer; 2020. p. 235–49.
Ali M, Ishqi HM, Husain Q. Enzyme engineering: Reshaping the biocatalytic functions. Biotechnol Bioeng. 2020;117:1877–94.
Chen L, Holmes M, Schaefer E, Mulchandani A, Ge X. Highly active spore biocatalyst by self-assembly of co-expressed anchoring scaffoldin and multimeric enzyme. Biotechnol Bioeng. 2018;115:557–64.
Jose J. Autodisplay: efficient bacterial surface display of recombinant proteins. Appl Microbiol Biotechnol. 2006;69:607–14.
Balderas Hernández VE, Salas-Montantes CJ, Barba-De la Rosa AP, De Leon-Rodriguez A. Autodisplay of an endo-1,4-β-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation. Enzyme Microb Technol. 2021;149:109834.
Gutiérrez-García AK, Alvarez-Guzmán CL, De Leon-Rodriguez A. Autodisplay of alpha amylase from Bacillus megaterium in E. coli for the bioconversion of starch into hydrogen, ethanol and succinic acid. Enzyme Microb Technol. 2020;134:109477.
Strätker K, Haidar S, Dubiel M, Estévez-Braun A, Jose J. Autodisplay of human PIP5K1α lipid kinase on Escherichia coli and inhibitor testing. Enzyme Microb Technol. 2021;143:109717.
Huo Z, Zhang N, Raza W, Huang X, Yong X, Liu Y, et al. Comparison of the spores of Paenibacillus polymyxa prepared at different temperatures. Biotechnol Lett. 2012;34:925–33.
Karava M, Gockel P, Kabisch J. Bacillus subtilis spore surface display of photodecarboxylase for the transformation of lipids to hydrocarbons. Sustain Energy Fuels. 2021;5:1727–33.
Chen H, Ullah J, Jia J. Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis. J Mol Microbiol Biotechnol. 2017;27:159–67.
Petrillo C, Castaldi S, Lanzilli M, Saggese A, Donadio G, Baccigalupi L, et al. The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis. Microb Cell Factories. 2020;19:185.
Kim J. Surface display of lipolytic enzyme, Lipase A and Lipase B of Bacillus subtilis on the Bacillus subtilis spore. Biotechnol Bioprocess Eng. 2017;22:462–8.
Hui Y, Cui Z, Sim S. Stress-Tolerant, Recyclable, and Renewable Biocatalyst Platform Enabled by Engineered Bacterial Spores. ACS Synth Biol. 2022;11:2857–68.
Knecht LD, Pasini P, Daunert S. Bacterial spores as platforms for bioanalytical and biomedical applications. Anal Bioanal Chem. 2011;400:977–89.
Isticato R, Ricca E. Spore Surface Display. In: The Bacterial Spore. John Wiley & Sons, Ltd; 2016. p. 349–66.
Csibra E, Renders M, Pinheiro VB. Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low-Affinity Ligands and Enzymes. ChemBioChem. 2020;21:2844–53.
Lin P, Yuan H, Du J, Liu K, Liu H, Wang T. Progress in research and application development of surface display technology using Bacillus subtilis spores. Appl Microbiol Biotechnol. 2020;104:2319–31.
Guoyan Z, Yingfeng A, Zabed HM, Qi G, Yang M, Jiao Y, et al. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. 2019;29:179–90.
Curtis D, Golomb BL, Ally D, Ficarra FA, Salamzade R, Traag BA. Paenibacillus-based endospore display platform, products and methods. 2021.
Langendries S, Goormachtig S. Paenibacillus polymyxa, a Jack of all trades. Environ Microbiol. 2021;23:5659–69.
Hinc K, Iwanicki A, Obuchowski M. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb Cell Factories. 2013;12:22.
Chen X, Zaro JL, Shen W-C. Fusion protein linkers: Property, design and functionality. Adv Drug Deliv Rev. 2013;65:1357–69.
van Rosmalen M, Krom M, Merkx M. Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins. Biochemistry. 2017. doi:10.1021/acs.biochem.7b00902.
Hosseini-Abari A, Kim B-G, Lee S-H, Emtiazi G, Kim W, Kim J-H. Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein. J Basic Microbiol. 2016;56:1331–7.
Kang SJ, Park EA, Lee DH, Hong KW. Comparison of the stability of eGFP displayed on the Bacillus subtilis spore surface using CotB and C-terminally truncated CotB proteins as an anchoring motif under extreme conditions. Appl Biol Chem. 2019;62:41.
Akram F, Mir AS, Haq I ul, Roohi A. An Appraisal on Prominent Industrial and Biotechnological Applications of Bacterial Lipases. Mol Biotechnol. 2023;65:521–43.
Kazlauskas RJ, Bornscheuer UT. Biotransformations with Lipases. In: Biotechnology. John Wiley & Sons, Ltd; 1998. p. 36–191.
Hama S, Noda H, Kondo A. How lipase technology contributes to evolution of biodiesel production using multiple feedstocks. Curr Opin Biotechnol. 2018;50:57–64.
Zhang Y, Ji F, Wang J, Pu Z, Jiang B, Bao Y. Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124. Extremophiles. 2018;22:287–300.
Pedreira T, Elfmann C, Stülke J. The current state of SubtiWiki, the database for the model organism Bacillus subtilis. Nucleic Acids Res. 2022;50:D875–82.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.
Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2021;50:D20–6.
Hwang B-Y, Pan J-G, Kim B-G, Kim J-H. Functional Display of Active Tetrameric β-Galactosidase Using Bacillus subtilis Spore Display System. J Nanosci Nanotechnol. 2013;13:2313–9.
Schilling C, Ciccone R, Sieber V, Schmid J. Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365. Metab Eng. 2020;61:381–8.
Meliawati M, Schilling C, Schmid J. Recent advances of Cas12a applications in bacteria. Appl Microbiol Biotechnol. 2021;105:2981–90.
Parks TD, Howard ED, Wolpert TJ, Arp DJ, Dougherty WG. Expression and Purification of a Recombinant Tobacco Etch Virus NIa Proteinase: Biochemical Analyses of the Full-Length and a Naturally Occurring Truncated Proteinase Form. Virology. 1995;210:194–201.
Martin N, Lombardía E, Altabe SG, de Mendoza D, Mansilla MC. A lipA (yutB) Mutant, Encoding Lipoic Acid Synthase, Provides Insight into the Interplay between Branched-Chain and Unsaturated Fatty Acid Biosynthesis in Bacillus subtilis. J Bacteriol. 2009;191:7447–55.
Babtie A, Tokuriki N, Hollfelder F. What makes an enzyme promiscuous? Curr Opin Chem Biol. 2010;14:200–7.
Dartois V, Baulard A, Schanck K, Colson C. Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168. Biochim Biophys Acta BBA - Gene Struct Expr. 1992;1131:253–60.
Noby N, Saeed H, Embaby AM, Pavlidis IV, Hussein A. Cloning, expression and characterization of cold active esterase (EstN7) from Bacillus cohnii strain N1: A novel member of family IV. Int J Biol Macromol. 2018;120:1247–55.
Jiang H, Zhang S, Gao H, Hu N. Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution. BMC Biotechnol. 2016;16:7.
Wei W, Ma J, Chen S-Q, Cai X-H, Wei D-Z. A novel cold-adapted type I pullulanase of Paenibacillus polymyxa Nws-pp2: in vivo functional expression and biochemical characterization of glucans hydrolyzates analysis. BMC Biotechnol. 2015;15:96.
Mattossovich R, Iacono R, Cangiano G, Cobucci-Ponzano B, Isticato R, Moracci M, et al. Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes. Microb Cell Factories. 2017;16:218.
Chen L, Mulchandani A, Ge X. Spore-displayed enzyme cascade with tunable stoichiometry. Biotechnol Prog. 2017;33:383–9.
Wang H, Wang Y, Yang R. Recent progress in Bacillus subtilis spore-surface display: concept, progress, and future. Appl Microbiol Biotechnol. 2017;101:933–49.
Rütering M, Cress BF, Schilling M, Rühmann B, Koffas MAG, Sieber V, et al. Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol. 2017;2:ysx007.
Washida M, Takahashi S, Ueda M, Tanaka A. Spacer-mediated display of active lipase on the yeast cell surface. Appl Microbiol Biotechnol. 2001;56:681–6.
Hartig SM. Basic Image Analysis and Manipulation in ImageJ. Curr Protoc Mol Biol. 2013;102:14.15.1-14.15.12.
Karava M. Development of a platform for immobilization of proteins based on Bacillus subtilis spores. Technische Universität Darmstadt; 2021.

No competing interests reported.

Supplementaryinformation.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Implementation of spore display in Paenibacillus polymyxa with different hydrolytic enzymes

Status:

Version 1

Abstract

Figures

Introduction

Results

Construction of strains and plasmids for the expression of fusion proteins on the spore surface

Identification of the GFP display via fluorescence microscopy and flow cytometry

Enzyme activity assays of the CotE-Lip3, CotE-LipA, and CotE-PnbA fusion proteins

Recyclability and reusability of the spore display

Discussion

Conclusion

Material and Methods

Bacterial Strain and Growth Conditions

Strain and Plasmid Construction

Sporulation and Purification

Fluorescence Microscopy and Image Analysis

Cytometric analysis

Hydrolysis activity assay

TEV-cleavage assay

SDS-Polyacrylamide-Gel electrophorese (SDS-PAGE)

Recycling of spore activity

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1