Spheroplasts preparation boosts the catalytic potential of a squalene-hopene cyclase

Squalene-hopene cyclases are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of squalene-hopene-cyclase spheroplasts. By removing the outer cell membrane, we produce stable and substrate-accessible biocatalysts. These spheroplasts exhibit up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability is also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a concept for the carrier-free immobilization of spheroplasts via crosslinking, crosslinked spheroplasts. The crosslinked spheroplasts maintain the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the squalene-hopene cyclases, but also to make biocatalytic processes even greener and more cost-efficient.


Supplementary Discussion
Supplementary After the biotransformations with immobilized cells on solid supports, a decrease of the substrate 1E/Z was detected while no product was found, especially when using the methacrylate microbeads which present a higher hydrophobicity ( Supplementary Fig. 1B). Therefore, we imagined that the substrate 1E/Z could either get stuck to the agarose/methacrylate support or trapped inside the cells. To decipher the spatial location of the substrate, we firstly extracted both substrate and product using two non-polar solvents such as cyclohexane and toluene. Only traces of product were detected in both cases, whereas the detection of substrate remained at 75±9% from the agarose microbeads and only 36±5% from the methacrylate microbeads (Supplementary Fig. 2A). Secondly, the substrate was incubated with agarose microbeads (without immobilized cells), methacrylate microbeads (without immobilized cells), and whole cells. This experiment revealed that most of the substrate (80%) is trapped inside the cells, although the stickiness to the agarose/methacrylate microbeads is a hurdle as well ( Supplementary Fig.   2B).
Supplementary Fig. 1. Immobilized whole cells on premade agarose (6BCL) and methacrylate (EP403/S) supports. A) Scheme of the activation of the microbeads with glyoxyl (Gx) groups and the immobilization of whole cells. The glyoxyl groups could interact with the lysines of the proteins on the cell membrane stablishing covalent bonds. B) Batch biotransformations with the immobilized whole cells. 200 mg of microbeads with immobilized cells (36 mgcells/gsupport) were added to 1 mL of 10 mM geranyl acetone 1E/Z, 1% DMSO, 0.2% SDS, and 20 mM citric acid buffer at pH 6.0. The reactions were incubated at 30 ºC for 24 h. The recovery (%) of substrate and product were calculated by using standard curves of the substrate and product. The differences on the substrate extraction could be attributed to cell permeabilization effects by the pH (Supplementary Fig. 3). It has been reported that buffer at more basic pH can increase the outer membrane permeability. 1  Fig. 4A-B) were employed for enzyme immobilization. 1 mg/g of protein could be immobilized on ECR8285 and HFA403, while 0.76 mg/g were immobilized on EP403/S ( Supplementary Fig. 4C). However, no enzyme activity was recovered after immobilization on ECR8285. Considering the substrate/product sticking issues observed with EP403 microbeads before, HFA403 was selected as support for AacSHC immobilization.

Supplementary
Supplementary Fig. 4. AacSHC immobilization on methacrylate microbeads. A) Scheme of the enzyme immobilization on EP403/S and ECR8285. Firstly, the protein (in red) is hydrophobically adsorbed due to a high ionic strength immobilization buffer. Then, a covalent linkage takes place between the nucleophilic groups (amino, hydroxyl and thiol) on the surface of the enzyme and the epoxy groups on the support. B) Scheme of the enzyme immobilization on HFA403. Firstly, the protein (in purple) is ionically bound to the amino groups on the support and then the covalent bond with the epoxy groups happens. C) Immobilization results. The offered protein loading was 1 mg/g.  Fig. 5A). In addition, we investigated the use of different molecules to block the remaining epoxy groups on the microbeads after enzyme immobilization, thus providing different microenvironments to the immobilized enzyme. Glycine (more hydrophilic environment) and ethylamine (more hydrophobic environment) are frequently used for this purpose and were tested in this work. 2,3 Both the enzymatic activity and the reusability of the immobilized enzyme showed better results when  Table 3. Continuous flow biotransformations with immobilized enzyme on methacrylate (HFA403) microbeads. Substrate solution: 10 mM geranyl acetone 1E/Z, 1% DMSO, and 10 mM citric acid buffer at pH 6.0. Reactor volume: 1.2 mL. Flow rate: 20-40 μL/min. SD <1%. For the biphasic reactions, the substrate solution (20 mM 1E/Z, 1% DMSO, 20 mM citric acid buffer pH 6.0) was mixed in a T-tube with the organic phase (1:1 ethyl acetate and cyclohexane). In case of recirculation, the collected solution with unreacted substrate was used to feed the flow reactor during 6 cycles increasing the overall contact time to 3 h (30 min R.T.) and 6 h (60 min R.T.). The conversion was calculated as the average of at least 3 column volumes. After the reaction was performed, the activity of the immobilized enzyme was tested in batch to confirm that the biphasic system had no influence on the enzyme activity.     Table 9

. Preliminary analysis of the costs comparing the use of whole cells, spheroplasts and CLS as biocatalysts at lab scale to synthesize the products 2E and 4. A)
Costs of the biocatalyst i n 10 mg l hiliz d . P i €/Kg f m igm Ald i h 16.08.2022, G m n . CL : linked spheroplasts. *For the cost of the lysozyme, the price of the larger format commercially available was considered (100 g) and extrapolated to 1 Kg. B) Costs of the reaction set-up. C) Overall costs calculated by considering both the biocatalyst preparation and the reaction set-. C l l d € /mgproduct were obtained from the results depicted in Fig.S9, considering the reusability of CLS (at least 4 reuses) showed in Fig. 4 Supplementary Table 10. Summary of all the immobilization strategies followed in this work. Conversions were calculated from a biotransformation in 1 mL using 2-10 mM of geranyl acetone 1E/Z after 24 h at 30ºC. PEI (polyethyleneimine), GA (glutaraldehyde), BDE (1,4-butanediol diglycidyl ether). n.t. (not tested).
[a] Immobilization yield of the free enzyme was calculated following equation 1: (1) in in h ini i l l i n-in in h n n f imm iliz i n in in h ini i l l i n x 100 [b] Immobilization yield for whole cells and spheroplasts was calculated following equation 2: (2) OD600 f h ini i l l i n -OD600 f h n n f imm iliz i n OD600 f h ini i l l i n x 100 [c] Retained activities were calculated following equation 3:  Supplementary Fig. 17. Gas chromatogram of squalene 3 to hopene 4. Supplementary Fig. 18. Gas chromatogram of E,E-farnesol 5 to drimenol 6. Supplementary Fig. 19. Gas chromatogram of E,E-farnesyl acetone 7 to sclareoloxide 8.

Supplementary Methods
Activity assessment of purified AacSHC. The activity of AacSHC (0.02 mg/mL or 0.1 mg/mL of free enzyme; 20 mg of immobilized enzyme) was assessed by monitoring the cyclization of 10 mM geranyl acetone in citric acid buffer (10 mM, pH 6.0) in a total reaction volume of 1 mL. 1 % DMSO was used as co-solvent. The reactions were incubated at 30 C for 20 h at 300 rpm. The samples were extracted with 1 mL of 1:1 ethyl acetate:cyclohexane.
After centrifugation (12,000 rpm, 10 min at 20 C), the solvent phase was analyzed by GC-FID.
Immobilization of AacSHC on commercially available methacrylate supports. was poured on a petri dish as a layer. After polymerization, the polyacrylamide hydrogel was cut into small pieces for a better handling washed with 20 mM citric buffer pH 6.0.
Thermolysis purification. 7 Spheroplasts or lyophilized cells were resuspended in 1 mL Lysis buffer (200 mM citrate, 0.1 % EDTA, pH 6.0) and incubated for 60 min at 70 °C. The cell suspension was centrifuged (14000 x g, 1 min) and the supernatant was kept for analysis. As the enzyme is membrane-bound, 1 mL 1%-CHAPS buffer (100 mM citrate, pH 6.0) was added to extract it from the cell pellet by shaking at room temperature for 1d, 600 rpm. After subsequent centrifugation (14000 x g, 1 min) the supernatant containing the AacSHC was transferred to a new tube followed by SDS-PAGE analysis and determination of enzyme concentration by using the Nanodrop 1000