catalysts Article Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability Josu López-Fernández , Maria Dolors Benaiges and Francisco Valero * Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; josu.lopez@uab.cat (J.L.-F.); mariadolors.Benaiges@uab.cat (M.D.B.) * Correspondence: francisco.valero@uab.cat; Tel.: +34-93-5811809 Abstract: Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methy- lotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP ). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final  activity and volumetric productivity more than six times higher than those obtained with proROL  from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1 ). The previous Citation: López-Fernández, J.; differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence Benaiges, M.D.; Valero, F. of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance Constitutive Expression in of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence Truncated Prosequence Improves enables constitutive proROL production, boosts bioprocess performance and provides a more stable Production and the Biocatalyst biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl Operational Stability. Catalysts 2021, butyrate) and transesterification (biodiesel). 11, 1192. https://doi.org/10.3390/ catal11101192 Keywords: Rhizopus oryzae lipase; Komagataella phaffii; prosequence; flavor; biodiesel; biocatalysis Academic Editor: Zheng Guo Received: 12 September 2021 1. Introduction Accepted: 29 September 2021 Biocatalysis has for some time been extensively used to obtain a variety of products in- Published: 30 September 2021 cluding polymers, fine and bulk chemicals, flavors and pharmaceutical intermediates [1,2]. Lipases (triacylglycerol hydrolases E.C. 3.1.1.3), which are the most widely used biocatalysts Publisher’s Note: MDPI stays neutral after proteases and amylases, catalyze a wide range of industrially interesting reactions with regard to jurisdictional claims in published maps and institutional affil- including esterification, transesterification and interesterification [3,4]. Specifically, Rhi- iations. zopus oryzae lipase (ROL) has been the subject of much study on account of a number of favorable traits such as 1,3-regioespecificity, high thermostability, tolerance of organic solvents, and the ability to produce flavors, biodiesel and structured lipids [5]. All lipases from the genus Rhizopus are synthesized as precursor forms sharing a common structure. Thus, ROL forms from a presequence of 26 amino acids that is followed by a prosequence Copyright: © 2021 by the authors. of 97 and a mature sequence of 269 [6]. Whereas the presequence is known to act as a signal Licensee MDPI, Basel, Switzerland. peptide, the specific functions of the prosequence are incompletely known but thought to This article is an open access article include increasing protein stability, reducing stress during recombinant protein expression, distributed under the terms and conditions of the Creative Commons and minimizing unfolded protein response (UPR) in rROL production—which has an Attribution (CC BY) license (https:// adverse impact on microbial growth [7–9]. Obtaining the mature form of the lipase (rROL) creativecommons.org/licenses/by/ entails removing the presequence and prosequence [10,11]. However, the native microor- 4.0/). ganism secretes a protein including the mature sequence and, attached to its N-terminal, Catalysts 2021, 11, 1192. https://doi.org/10.3390/catal11101192 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, 1192 2 of 15 the 28 C-terminal amino acids of the prosequence (proROL), which are subsequently pro- teolyzed by extracellular proteases. The 28 amino acids by themselves seemingly suffice for the previous positive traits of the whole prosequence to occur and are thus important with a view to developing a potentially improved ROL-based biocatalyst [7]. The industrial use of enzymes is often hampered by their high cost and also, often, by their low thermostability and poor tolerance of organic solvents. These shortcomings are usually circumvented by immobilization or heterologous production [12,13]. Immobiliza- tion facilitates the recycling of biocatalysts and usually increases their stability. Enzymes are typically immobilized by binding to a support, entrapment or cross-linking [14,15]. ROL can be effectively immobilized in these three ways (e.g., by adsorption [16], covalent bind- ing [17], entrapment into alginate beads [18] or formation of cross-linked aggregates [19]). Immobilized biocatalysts have proved highly suitable for industrial-scale applications such as biodiesel and flavor production [5]. Heterologous production of enzymes is a more efficient and economical method than using native microorganisms [20]. ROL has been obtained to date from the well-known cell factories Escherichia coli, Saccharomyces cerevisiae and Komagataella phaffii (Pichia pastoris). In E. coli, however, the presence of three disulfide bonds in the protein, and the absence of suitable enzymes for maturation of fungal lipases, leads to the production of inactive lipase [21]. Nevertheless, by using the E. coli Origami (DE3) strain and pET-11d expression system, Di Lorenzo et al. succeeded in obtaining two ROL forms containing the mature sequence alone and the whole prosequence as well [22]. However, even if the soluble and active production of lipases was achieved, the fact that the enzymes were intracellularly produced—therefore requiring further purification steps—as well as the lack of protein processing mechanisms, since the lipase that contained the complete prosequence was not properly hydrolyzed, promoted the assessment of the yeasts S. cerevisiae and K. phaffii as potential sources for this enzyme. The different proteolytic processing imposed by Kex2-like protease in S. cerevisiae enabled the production of two lipase forms by the gene encoding the whole prosequence linked to the mature sequence (w-proROL-gene), unlike observed with E. coli; however, no activity was detected when only the mature sequence gene (rROL-gene) was expressed, which highlights the importance of the prosequence for efficient production of the lipase [5]. With the methylotrophic yeast K. phaffii, only the cleaved form of ROL was obtained after w-proROL-gene expression, which underlies the increased activity of Kex2-like protease in this microorganism [23]. In addition, unlike S. cerevisiae, K. phaffii expressed rROL-gene [24], which led to K. phaffii being deemed the best host for ROL heterologous production. This yeast has been reported to possess a number of advantages such as the absence of endogenous esterases or lipases, and the ability to secrete heterologous proteins, enact eukaryotic post-translational modifications and grow at high cell densities (ca. 100 g L−1 dry cell weight) in some media [25,26]. The alcohol oxidase 1 promoter (PAOX1 ) has been extensively used to express ROL in K. phaffii. This promoter is strongly induced by methanol but repressed by glucose and glyc- erol. Although PAOX1 allows large amounts of protein to be obtained, the use of methanol increases oxygen requirements and heat production—and raises production costs through the need to store and handle methanol properly [27,28]. These drawbacks have prompted the use of methanol-independent promoters such as the constitutive glyceraldehyde-3- phosphate dehydrogenase promoter (PGAP ) from central carbon metabolism [29]. Despite the constitutive nature of PGAP , its strong expression capacity depends on the carbon source and growth rate of the particular culture [30,31]. Although the expression of ROL has been already reported under this constitutive promoter, w-proROL-gene, which encodes both the prosequence and the mature sequence, is needed to alleviate the adverse effects of producing the mature sequence of ROL under PGAP [32], as this lipase form is harmful to the host cell [10]. In fact, rROL-gene expression has only been accomplished under the inducible PAOX1 [7,33]—inducible promoters are less troublesome than constitutive promoters in this respect [34]. Catalysts 2021, 11, 1192 3 of 15 The primary aim of this work was to express ROL under the constitutive promoter PGAP by using the 28 amino acids of its prosequence fused to the mature sequence to enable a methanol-independent and more environmentally friendly bioprocess. The alleged positive traits of the 28 C-terminal amino acids of the prosequence were assessed in terms of reducing the impact on host strain growth, increasing bioprocess productivity, improving the stability of the immobilized biocatalyst against organic solvents and boosting its operational stability during biodiesel and ethyl butyrate (pineapple flavor) production. 2. Results and Discussion 2.1. Batch and Fed-Batch Bioprocess Strategies. Lipase Production under Inducible PAOX1 and Constitutive PGAP The fact that some of the positive traits of the whole prosequence have also been identified with truncated sequences [21,35], led us to transform K. phaffii with proROL PGAP -plasmid, rROL PGAP -plasmid and the empty plasmid—blank—to investigate the role of the C-terminal 28 amino acids of the prosequence. Three transformation runs with each plasmid produced 252 colonies with the blank and 21 with proROL PGAP -plasmid but none with rROL PGAP -plasmid. Based on this outcome, the ROL mature sequence must somehow hinder constitutive expression in K. phaffii. In fact, these results are consistent with those of previous work where production of the ROL mature sequence caused cell lysis in E. coli owing to its phospholipase activity but no lysis when 28 C-terminal amino acids in the prosequence were expressed together with the mature sequence, although noxious effects were still observed as cell growth was inhibited [10]. Consequently, the truncated prosequence of ROL (28 C-terminal amino acids) can be assumed to alleviate the deleterious effects of the lipase and enable production of viable clones of K. phaffii as a result. Viable clones expressing proROL were identified by colony PCR and a pool of positive colonies was selected as described in Section 3.2, the most productive clone being chosen for further testing. Single plasmid integration in the proROL PGAP -strain clone was confirmed by ddPCR and duplicate batch cultures were run with glycerol as carbon source. Key production and growth parameters were compared with reported values for proROL PAOX1 -strain and rROL PAOX1 -strain grown in methanol [7] (Table 1). The greatest differences between proROL PAOX1 -strain and proROL PGAP -strain were those in final activity and volumetric productivity, which were 6.5 times higher in the latter. Additionally, qp was 2.2 times higher with proROL PGAP -strain than it was with proROL PAOX1 -strain. However, Y(P/X) was greater with the latter than it was with the former. In line with these results, specific productivities were very similar with both strains. In fact, the increased Y(P/X) value obtained with proROL PAOX1 -strain was offset by the increased specific growth rate of proROL PGAP -strain, which reduced bioprocess operation time. Table 1. Results obtained in batch tests involving proROL PAOX1 -strain and rROL PAOX1 -strain in 10 g MeOH L−1 , and proROL PGAP -strain in 40 g glycerol L−1 . Parameter proROL PAOX1 1 proROL PGAP rROL PAOX1 1 Final activity (AU mL−1 ) 12.38 74.71 10.51 Y(P/X) (total AU total gX −1 ) 5017 4273 3753 µ (h−1 ) 0.073 0.22 0.045 qp (AU gX −1 h−1 ) 391 874 168 Specific productivity (AU gX −1 h−1 ) 195 192 139 Volumetric productivity (AU L−1 h−1 ) 462 3367 389 1 Data obtained from [7]. The truncated prosequence minimized the harmful effects of mature ROL production on K. phaffii growth, no matter the promoter employed (Table 1). In fact, growth rates with proROL-producing strains were similar to those of wild-type strain under both glycerol (0.22 h−1 with proROL PGAP -strain and 0.25 h−1 with the wild-type strain) and methanol (0.073 h−1 with proROL PAOX1 -strain and 0.09 h−1 with the wild-type strain). By contrast, Catalysts 2021, 11, 1192 4 of 15 the growth rate of rROL PAOX1 -strain in methanol was markedly lower (0.045 h−1 ). These results suggest that K. phaffii is an effective cell factory for ROL production; as unlike above described with E. coli, in which proROL expression inhibited cell growth [10]. proROL PGAP -strain was also grown in fed-batch cultures, using a pre-programmed exponential glucose feeding rate to maintain a constant specific growth rate of 0.045 h−1 . Various key bioprocess parameters for proROL PGAP -strain were determined for compar- ison with previously reported results (Table 2) by using proROL PAOX1 -strain and the methanol limited fed-batch (MLFB) strategy at a constant specific growth rate, and the methanol non-limited fed-batch (MNLFB) strategy at a constant methanol concentration of 3 g L−1 in the culture broth throughout the induction stage. Based on previously obtained evidence, the latter is the more suitable strategy with this production system [7]. Table 2. Results obtained with proROL PGAP -strain under carbon-limited fed-batch conditions, and also with proROL PAOX1 -strain under either methanol-limited fed-batch (MLFB) conditions at a preset specific growth rate of 0.045 h−1 or methanol non-limited fed-batch (MNLFB) conditions at a constant methanol concentration of 3 g L−1 . Carbon-Limited Fed-Batch MNLFB µset-point = 0.045 h−1 3 g L−1 1 proROL Parameter proROL PAOX 1 proROL PGAP PAOX1 1 Final activity (AU mL−1 ) 147 341 358 Y(P/X) (total AU total gX −1 ) 1908 6789 4972 Estimated µ (h−1 ) 0.038 0.045 0.065 qp (AU gX −1 h−1 ) 68.5 479 308 Specific productivity (AU gX −1 h−1 ) 44 156 99 Volumetric productivity (AU L−1 h−1 ) 2782 7881 7160 1 Data obtained from [7]. As in the batch cultures, final activity (Figure 1) and volumetric productivity (Table 2) with proROL PGAP -strain and the fed-batch strategy were higher than with proROL PAOX1 - strain grown under carbon-limited fed-batch conditions at a similar specific growth rate [7]. However, Y(P/X) was greater with proROL PGAP -strain than it was with proROL PAOX1 - strain, contrary to what was observed during batch cultures. This outcome is consis- tent with the results of previous work were the MLFB strategy was reported to perform poorly [36]. Interestingly, Y(P/X) under MNLFB was 1.5 times higher with proROL PGAP -strain than it was with proROL PAOX1 -strain. However, final activity and volumetric productivity were similar with both strains. Thus, the bioprocess parameters for proROL PGAP -strain grown at specific rate of 0.045 h−1 on glucose were similar to those provided by proROL PAOX1 -strain with the best strategy devised so far. Therefore, this strategy not only enables methanol-free proROL production but also lends itself readily to bioprocess optimization with proROL PGAP -strain. Catalysts 2021,2021, Catalysts 11, x11, FOR1192PEER REVIEW 5 of 15 Figure 1.1.Time Figure Time course of lipase course activity of lipase in carbon activity in limited carbonfed-batch limited cultures fed-batchof proROL PGAP cultures of-strain proROL PGAP and proROL PAOX1 -strain at a preset specific growth rate of 0.045 h−1 (dashed line) and proROL PAOX1-strain at a preset specific growth rate of 0.045 h−1 (dashed MNLFB and with line) and with (solid line). Strains: proROL PAOX1 (circles) and proROL PGAP (triangles). MNLFB (solid line). Strains: proROL PAOX1 (circles) and proROL PGAP (triangles). 2.2. N-Terminal Amino Acids in proROL, rROL and proROLm Free Enzymes Interestingly, Previous Y(P/X) under results obtained MNLFBmolecular by SDS-PAGE was 1.5weight times analysis highersuggested with proROL PGAP-stra selective itproteolysis was with proROL of the PAOX1 N-terminal 28-strain. However, amino acids final activity in the truncated andofvolumetric prosequence proROL by produ were similar with both strains. Thus, the bioprocess parameters for whose effect of proteases [7]. Similar results were also obtained with R. niveus lipase, proROL PGA N-terminal is identical with that of ROL [37]. Therefore, these 28 amino acids seem to grown at specific rate of 0.045h on glucose were similar to those provided by p −1 be naturally designed for removal to obtain the lipase mature sequence (rROL). proROL Pbehaved AOX1-strain with the best strategy devised so far. Therefore, this strategy not only identically here. The qualitative information of proteolysis obtained by SDS-PAGE methanol-free proROLby technique was confirmed production N-terminal but also lends sequence itself analysis, readily which to bioprocess involved automated optim with proROL Edman’s PGAP-strain. degradation of proROLm (proteolyzed proROL, see Section 3.5), and comparison with proROL and rROL N-terminal (Table 3). The 28 amino acids in the prosequence were confirmed to be primarily hydrolyzed by proteases in order to convert proROL into the 2.2. N-Terminal Amino Acids in proROL, rROL and proROLm Free Enzymes lipase mature sequence (rROL). However, the expected proROLm sequence, which should have Previous results been identical withobtained by SDS-PAGE that of rROL, molecular did not materialize becauseweight the firstanalysis serine wassuggeste removed. tive Based onofstudies proteolysis done with the the N-terminal 28bioinformatic amino acids tool inExpasy, the digestion the truncated pattern prosequence of p could be ascribed to AspN and AspGluN endoproteases. by effect of proteases [7]. Similar results were also obtained with R. niveus lipase, N-terminal Table 3. Expectedis identical sequence forwith that of based the N-terminal ROLon[37]. Therefore, the cloned sequencethese 28 amino and actual sequenceacids as seem naturally determined designed for removal by Edman’s degradation to obtain the lipase mature sequence (rROL). proR analysis. haved identically here. The qualitative information of proteolysis obtained by SDS Enzyme Expected Sequence Actual Sequence technique was confirmed by N-terminal sequence analysis, which involved auto proROL DDNLVG EADDNL Edman’s degradation of proROLm (proteolyzed proROL, see Section 3.5), and co proROLm SDGGKVV DGGKVV son with proROL and rROL N-terminal (Table 3). The 28 amino acids in the prose rROL 1 SDGGKVV EAEFSDGGKVVAA were confirmed to be primarily hydrolyzed by proteases in order to convert proRO 1 Data obtained from [38]. the lipase mature sequence (rROL). However, the expected proROLm sequence should have been Regarding proROL identical withas N-terminal, that of rROL, previously did not reported formaterialize rROL [38], the because plasmidthe firs used for heterologous production of the lipase in K. phaffii was removed. Based on studies done with the bioinformatic tool Expasy, left the last two amino acids the di of the alpha-factor sequence of S. cerevisiae (EA) in the resulting protein. The presence of pattern could be ascribed to AspN and AspGluN endoproteases. these amino acids is important because the theoretical isoelectric point (pI) for proROL as calculated with Expasy bioinformatic tool switches from 6.32 to 6.08, which explains Table 3. Expected sequence for the N-terminal based on the cloned sequence and actual seq as determined by Edman’s degradation analysis. Enzyme Expected Sequence Actual Sequence these amino acids is important because the theoretical isoelectric point (pI calculated with Expasy bioinformatic tool switches from 6.32 to 6.08, whic viously encountered turbidity issues in proROL strain fermentations at p near the pI level leading to enzyme denaturation [7]. In addition, unlike in Catalysts 2021, 11, 1192 6 of 15 following two amino acids, EF—an unwanted sequence coming from the r were not found because plasmid restriction sites were upgraded. Then, in EA amino previously acids, turbidity encountered proROL N-terminal issues showed in proROL strain the same fermentations at pHsequence 5.5—a value as nat Rhizopus near oryzae the pI level (indicated leading to enzymeindenaturation Table 3 as[7].the In expected sequence) addition, unlike in rROL [39]. [38], This the following two amino acids, EF—an unwanted sequence coming explain that in previous work, proROL and the native ROL showed similarfrom the restriction site—were not found because plasmid restriction sites were upgraded. Then, in addition toificity the EAtowards p-nitrophenol amino acids, proROL N-terminalesters of different showed chain length the same sequence [7].lipase as native from Rhizopus oryzae (indicated in Table 3 as the expected sequence) [39]. This outcome might 2.3. explain thatof Stability inEB-proROL previous work,and proROL and the native EB-rROL in theROL showedof Presence similar substrate Organic Solvents specificity towards p-nitrophenol esters of different chain length [7]. For industrial use, biocatalysts must be stable enough in the organi 2.3. Stability cally used of EB-proROL [40]. ROLandhas EB-rROL beenin deemed the Presencetolerant of Organic Solvents of non-aqueous solvents For industrial use, biocatalysts must be stable enough in the organic solvents typically because proROL is a more stable enzyme than rROL by virtue of its truncate used [40]. ROL has been deemed tolerant of non-aqueous solvents [41]. However, because [21], in proROL is athis morework we compared stable enzyme than rROL the stability by virtue of bothprosequence of its truncated lipases covalently [21], in im a support this containing work we compared epoxyofand the stability butyl covalently both lipases functional groups onto immobilized (EB)a in solvents sp support containing epoxy and butyl functional groups (EB) in solvents spanning a wide range of range of Log p values (Figure 2). Log p values (Figure 2). Figure Figure 2. Relative 2. Relative initial rate initial reaction reaction rate butyrate (%) of ethyl (%) of ethyl butyrate synthesis synthesis after incubation of theafter incubati biocat- alysts for 24 h in the following solvents: tetrahydrofuran (THF), anisole, limonene, alysts for 24 h in the following solvents: tetrahydrofuran (THF), anisole, limonene cyclohexane, heptane and isooctane. The Log p values for each solvent are shown under its name. EB-rROL (black). heptane and isooctane. The Log p values for each solvent are shown under its nam EB-proROL (striped grey). The initial reaction rate for each non-incubated biocatalyst was taken to (black). EB-proROL (striped grey). The initial reaction rate for each non-incubated be 100%. taken to be 100%. The presence of the truncated prosequence had a positive effect on EB-proROL stability, which exceeded that of EB-rROL in all cases. In fact, EB-proROL was 2.5 times more stable The presence than EB-rROL of the (THF), in tetrahydrofuran truncated prosequence the most had extreme case. In a positive anisole effect on E and limonene, bility, two which solvents withexceeded that for a high potential of EB-rROL use in greenin all cases. chemistry [42]In fact, and EB-proROL medium Log p was values, EB-proROL was about 30% more stable than EB-rROL. Log p stable than EB-rROL in tetrahydrofuran (THF), the most extreme case. In a had a marked effect on biocatalyst stability, decreasing values—thus, more miscible solvents with water, such asonene, two solvents THF—leading with to increasing a high reduction of potential stability duefor use to the in green removal chemistry of structural water [42] an p values, from EB-proROL the enzymes, which iswas about required 30% more to maintain theirstable than EB-rROL. 3D structure and activity Log [43]. p had Obviously, the adverse effects of low Log p values on proROL were less on biocatalyst stability, decreasing values—thus, more miscible solvents w marked than those on rROL. as THF—leading to increasing reduction of stability due to the removal of s from the enzymes, which is required to maintain their 3D structure an Catalysts 2021, 11, 1192 7 of 15 The results for EB-rROL in heptane and isooctane were compared with previously reported values for rROL immobilized onto various supports (EP100, Eupergit® CM and octadecyl-Sepabeads). This way, immobilization onto the EB support obtained from Purolite® was found to increase the stability of rROL [44]. 2.4. Transesterification and Esterification Reactions. Initial Reaction Rate and Operational Stability rROL and proROL free lipases were covalently immobilized onto a Purolite® D6307 support containing epoxide and butyl functional groups (EB) to elucidate the influence of the 28 amino acids in the lipase on the initial reaction rate and operational stability of the biocatalyst during biodiesel and ethyl butyrate production. 2.4.1. Biodiesel Production The enzymatic production of biodiesel has several advantages in terms of process sustainability over its chemical production. However, only exceptionally stable biocatalysts allow cost-effective production [45,46]. This led us to assess the potential of the truncated prosequence of ROL for increasing the operational stability of the resulting biocatalyst under enzymatic biodiesel production conditions and its influence on the initial trans- esterification rate. In addition, due to 1,3-regiospecificity of Rhizopus oryzae lipase, the stoichiometric amount of alcohol (viz., a 2:1 alcohol-to-oil mole ratio) was employed to avoid the presence of too high alcohol concentration in reaction medium and hence, further adverse effects on the operational stability. Besides, ROL regioespecificity avoids glycerol formation during transesterification—a low value compound that might hinder enzyme performance [47]—and gives 2-monoacylglyceride a product with a high added value for the cosmetics and food industries [48]. No differences in initial reaction rate were observed between the two biocatalysts studied: EB-rROL and EB-proROL (Table 4). Therefore, the presence of the 28 amino acids in proROL had no influence on this parameter even though previous results suggested that its being close to the “lid region” in the 3D structure might interfere with interfacial activation and substrate–enzyme interaction processes [49,50]. Table 4. EB-rROL and EB-proROL initial transesterification and esterification rates, productivity and half-lives as calculated by using the best-fitting deactivation model. Initial Rate Productivity Reaction Biocatalyst (µmol Product (µmol Product Half-Life (h) mL−1 min−1 ) min−1 ) EB-proROL 27.2 39.43 498 Transesterification EB-rROL 25.1 34.17 102 EB-proROL 308 4.74 70 Esterification EB-rROL 294 3.10 30 As regards operational stability, Figure 3A shows the relative yield obtained in con- secutive transesterification reaction cycles with each immobilized lipase. After 8 cycles, EB-proROL exhibited the greatest operational stability, with a relative yield exceeding 90% of the initial value and that of EB-rROL (70%) by 25%. These results testify to the influence of biocatalyst stability on productivity and half-life (Table 4). In fact, after 8 reaction cycles EB-proROL exhibited 15% higher productivity than EB-rROL. Half-lives were calculated by fitting the experimental relative yields with the deactivation mod- els described in Section 3.8. The results obtained with EB-proROL fitted Equation (2) (Equation (2), a two-component first-order exponential decay model (2)) more closely than they fitted Equation (1) (Equation (1), a first-order exponential decay model (1)); thus, R2 was 0.844 with Equation (2) and 0.7389 with Equation (1). On the other hand, the results for EB-rROL fitted both models almost identically well, R2 being 0.977 with Equation (1) fitted Equation (1) (Equation (1), a first-order exponential decay model (1)); thus, R2 was Catalysts 2021, 11, 1192 0.844 with Equation (2) and 0.7389 with Equation (1). On the other hand, the results 8 of 15 for EB-rROL fitted both models almost identically well, R being 0.977 with Equation (1) and 2 0.991 with Equation (2). The best model for each biocatalyst (Equation (2)) was used to calculate the corresponding half-life. As can be seen from Table 4, t1/2 was almost 5 times and 0.991 with Equation (2). The best model for each biocatalyst (Equation (2)) was used greater to calculateEB-proROL with than ithalf-life. the corresponding was withAsEB-rROL. can be seen Similar results4, were from Table previously t1/2 was almost ob- tained with 5 times a whole-cell greater ROL biocatalyst with EB-proROL than it was (456 with h) [51], rROL EB-rROL. immobilized Similar onto Purolite® results were previously Lifetech obtainedECR1030M TM (579 h) with a whole-cell andbiocatalyst ROL IRA-96 (381 (456h) h)[52]. [51], By rROLcontrast, rROL onto immobilized Purolite® onto immobilized LifetechTM AP1090M andECR1030M Lewatit VP (579OCh) 1600 and IRA-96 had a (381 h) [52].shorter markedly By contrast, rROL half-life immobilized (270 and 113 h, re- onto AP1090M spectively). and Lewatit Interestingly, rROLVP OC 1600 had a markedly immobilized shorter onto Lifetech TM half-life ECR8285M—a(270 and biocatalyst 113 h, respectively). Interestingly, rROL immobilized onto Lifetech TM ECR8285M—a biocatalyst identical with EB-rROL— had a much shorter half-life: 16 h [52]. Since the only difference identical with EB-rROL— had a much shorter half-life: 16 h [52]. Since the only difference was that in reaction conditions, these results testify to the importance of reaction condi- was that in reaction conditions, these results testify to the importance of reaction conditions tions in biocatalyst operational stability. in biocatalyst operational stability. FigureFigure 3. (A)3.Relative yield (A) Relative of of yield consecutive consecutive transesterification runs transesterification runs withwith eacheach biocatalyst biocatalyst as obtained as obtained by following by following the 1-pulsethe 1- pulse ethanol ethanoladdition addition procedure. procedure. (B)(B) Relative Relative yieldyield of consecutive of consecutive esterification esterification reactions reactions of each biocatalyst of each biocatalyst as obtainedaswith obtained with butyric acidand butyric acid andethanol ethanolasas substrates. substrates. EB-rROL EB-rROL (black) (black) and EB-proROL and EB-proROL (striped (striped grey). grey). The The final final yield yield of the ofrun first thewas first run was taken takento to be be 100%. 100%. 2.4.2. 2.4.2. Ethyl Ethyl ButyrateProduction Butyrate Production Short-chainesters Short-chain estersare are arousing arousing increasing increasinginterest interest in in sectors such sectors as agri-food such and and as agri-food cosmetic production by virtue of their pleasant sensory attributes [53]. One such ester is cosmetic production by virtue of their pleasant sensory attributes [53]. One such ester is ethyl butyrate, which possesses a characteristic strong pineapple flavor and can be obtained ethyl butyrate, which possesses a characteristic strong pineapple flavor and can be ob- by esterifying butyric acid with ethanol. Although ROL has been successfully used to tained by esterifying synthesize butyricprevious ethyl butyrate, acid with workethanol. showed Although that a moreROL has been stable lipasesuccessfully is needed to used to synthesize ethyl butyrate, previous work showed that a more stable prevent deactivation by both the acid and the alcohol [54]. This led us to test immobilized lipase is needed to prevent proROL deactivation by both with here for comparison the acid rROL and the alcohol in order to assess[54]. the This led us of significance to the testtruncated immobilized prosequence proROL here for with a view to improving comparison with rROLthe operational in order stability to assess of the biocatalyst the significance of theintruncated this bioprocess and examining its influence on the initial reaction rate. prosequence with a view to improving the operational stability of the biocatalyst in this bioprocessAs in biodiesel and examiningproduction, the truncated its influence prosequence on the initial reaction had no effect on the initial rate. esterification rate. In fact, both biocatalysts led to an identical value in the first reaction As in biodiesel production, the truncated prosequence had no effect on the initial cycle (Table 4, Figure 3B). On the other hand, the truncated prosequence strongly affected esterification rate. In fact, both biocatalysts led to an identical value in the first reaction the operational stability of the biocatalyst during ethyl butyrate synthesis—even to a cycle (Table greater 4, Figure extent than in3B). On theproduction. biodiesel other hand, the truncated Thus, prosequence after 5 reaction strongly led cycles, EB-proROL affected thetooperational a 3 times higher relative yield than did EB-rROL (Figure 3B). As a result, productivity to a stability of the biocatalyst during ethyl butyrate synthesis—even greater with extent EB-proROLthan was in biodiesel 35% higher production. than it was Thus, with after 5 reaction EB-rROL. cycles, EB-proROL The half-lives of the two led to abiocatalysts 3 times higherwere relative calculated yield than did by fitting EB-rROL the relative (Figure yield results3B). As a result, to Equations (1)productivity and (2). Both fitted Equation (2) better than they fitted Equation (1) (EB-rROL R 2 = 0.9004 and with EB-proROL was 35% higher than it was with EB-rROL. The half-lives of the two bi- 2 = 0.9694). Besides, EB-proROL proved more stable (Table 4): its half-life EB-proROL ocatalysts wereRcalculated by fitting the relative yield results to Equations (1) and (2). Both was 2.5 times greater. Therefore, EB-proROL stands as a promising biocatalyst to avoid fitted Equation (2) better than they fitted Equation (1) (EB-rROL R2 = 0.9004 and EB-pro- deactivation by ethanol or butyric acid during esterification. ROL R = 0.9694). Besides, EB-proROL proved more stable (Table 4): its half-life was 2.5 2 times greater. Therefore, EB-proROL stands as a promising biocatalyst to avoid deactiva- tion by ethanol or butyric acid during esterification. Catalysts 2021, 11, 1192 9 of 15 3. Materials and Methods 3.1. Chemicals Olive pomace oil (alperujo) was a gift from Professor Eulogio Castro (University of Jaen, Spain). The immobilization support (polymethacrylate-based matrix D6307 containing epoxy and butyl functional groups) was kindly donated by Purolite® (King of Prussia, PA, USA). Heptane, methanol and ethanol were purchased from Panreac (Barcelona, Spain). Butyric acid, ethyl butyrate, standards of methyl/ethyl palmitate, methyl/ethyl stearate, methyl/ethyl oleate, methyl/ethyl linoleate, methyl linoleate and all other reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA). 3.2. Strains and Plasmids Four different plasmids were used, namely: two pPICZαA plasmids containing the mature sequence of Rhizopus oryzae lipase (rROL PAOX1 -plasmid), and a derivative thereof containing the 28 C-terminal amino acids of the prosequence of the native lipase in the N-terminal of rROL (proROL PAOX1 -plasmid), the pair expressed under the inducible promoter of alcohol oxidase 1 (PAOX1 ) [7]. In both plasmids, PAOX1 was replaced with the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP ) from pGAPZαA plasmid. Because exchanged fragments natively contained flanking sequences of BglII and XhoI restrictions sites, no unwanted nucleotides remained in the anew formed plasmids (rROL PGAP -plasmid and proROL PGAP -plasmid). More detailed information about the plasmids is provided in the Supplementary Material. Pichia pastoris X33 strains were transformed by electroporing 100 ng of previously linearized proROL PGAP -plasmid and rROL PGAP -plasmid. A blank consisting of empty pPICZαA plasmid with the promoter exchanged was additionally used as a control. The resulting colonies were re-streaked twice on YPD-Zeo plates to avoid population mixing, and five isolated colonies screened as described elsewhere [55], the most representative colony in each run being selected for further study. Single-gene copies were confirmed by digital droplet PCR [56]. 3.3. Batch Cultures An inoculum containing 100 mL of YPG medium plus zeocin to a final concentration of 100 µg mL−1 was grown in a 1 L baffled shake flask at 30 ◦ C that was stirred at 150 rpm for 24 h [57]. Then, a sample was centrifuged and resuspended in 100 mL of sterile water to inoculate the bioreactor to a final optical density (OD600 ) in 1 L of culture medium containing glycerol as sole carbon source [57]. Batch cultures were run in a 2 L bioreactor from Applikon Biotechnology (Delft, The Netherlands). The growing conditions were adapted from reported recommendations [58]. Thus, the temperature was kept at 30 ◦ C, and pH 5 was maintained by adding 15% (v/v) NH4 OH as required. The oxygen concentration was set at 25% and controlled by cascade stirring at 500–1200 rpm with constant aeration at 1 vvm. The end of each batch run was detected as an abrupt increase in dissolved oxygen (DO) concentration [58]. All tests were performed in duplicate. 3.4. Fed-Batch Cultures The inoculum was grown as described in the previous section and added to the biore- actor at a final OD600 of 1.5 in 2 L of the same culture medium used in the batch tests. Fed- batch cultures were run in a 5 L Biostat B bioreactor from Sartorius (Guxhagen, Germany). Batch and fed-batch stages were run by following previously reported procedures that were modified as described in the previous section [58]. The fed-batch stage was conducted with glucose as sole carbon source, using a carbon-limited feeding strategy and a preset exponential feeding rate intended to maintain a constant specific growth rate of 0.045 h−1 . All equations used to calculate yields and rates are based on mass balances in batch and fed-batch operations, and can be found elsewhere [59]. Catalysts 2021, 11, 1192 10 of 15 3.5. Lipase Production and Activity Assessment Fed-batch bioprocesses were followed by centrifugation, microfiltration, ultrafiltration and lyophilization of the culture broth to remove biomass in order to concentrate rROL and proROL [60]. Due to the impossibility of producing rROL under the constitutive promoter (PGAP ), the mature sequence lipase rROL was obtained from Komagataella phaffii strains producing the protein under the methanol inducible promoter (PAOX1 ) [7]. proROL derived lipase (proROLm) was obtained by action of proteases [7]. Then, centrifu- gation and passage through 0.2 µm filters was performed to remove unwanted contaminants. Lipase activity was determined on a Cary 300 spectrophotometer from Varian (Mul- grave, VIC, Australia), using the 11821792 lipase colorimetric kit from Roche (Mannheim, Germany) in 200 mM Tris-HCl buffer at pH 7.25 at 30 ◦ C. Measurements were made in triplicate at 580 nm [61]. 3.6. N-Terminal Analysis The N-terminal sequences of proROL, proROLm and rROL were determined by automated Edman’s degradation as described elsewhere [62]. The resulting sequences were further examined by using the Protein BLAST tool from the National Centre for Biotechnology Information (NCBI). 3.7. Lipase Immobilization proROL and rROL were immobilized onto a pretreated polymethacrylate matrix support (Purolite® D6307 with epoxide and butyl functional groups) by following a slightly modified version of a previously reported procedure [63]. Thus, epoxide functional groups were pretreated by incubating 1 g of support with 1 M ethylenediamine solution pH 10 under orbital stirring during 4 h at 60 ◦ C. Then, rinsed with distilled water by vacuum filtration and incubated in a 2.5% w/v glutaraldehyde solution at pH 8 on a roller during 2 h at room temperature. Finally, after rinsing the support, glutaraldehyde-treated support was incubated with a 600 AU mL−1 lipase solution at 4 ◦ C for 42 h. The biocatalysts thus obtained (EB-rROL and EB-proROL) were vacuum-filtered, dried on silica gel and stored at −20 ◦ C until use. The specific activity of the immobilized derivative was calculated by dividing the difference between final blank and final supernatant activity into the final dry weight of biocatalyst. 3.8. Transesterification and Esterification Reactions All transesterification and esterification reactions were conducted in 10 mL closed vials that were placed in a KS 400 incubator from IKA Werke (Staufen, Germany) at 30 ◦ C under orbital stirring at 350 rpm. Samples were periodically taken for reaction performance assessment by gas chromatography. Biodiesel transesterification reactions were run by using a total amount of biocatalyst corresponding to 32,000 AU and 8 g of olive pomace oil for 6 h. The stoichiometric amount of alcohol (2:1 alcohol to oil mole ratio) needed to obtain the theoretical maximum yield (66% based on the 1,3-regiospecificity of ROL) was added at the beginning of the reaction by following the already described 1-pulse ethanol addition procedure [64]. Operational stability was measured by allowing the biocatalysts to settle in the vial bottom and remov- ing the medium after each reaction run. Then, all reaction components were prepared for subsequent transesterification cycles [64]. The initial transesterification rate was determined by using a previously reported method [63]. Briefly, 1.56% (w/w) of methanol was added to olive pomace oil—the stoi- chiometric amount needed to obtain a yield of ca. 14%—and samples were withdrawn for analysis at regular intervals over the first 90 min of reaction. Ethyl butyrate production reactions were conducted by using 100 mM butyric acid and an acid:alcohol mole ratio of 1.25:1 to a final heptane volume of 8 mL. All substrates and solvents were dried with UOP type 3 molecular sieves from Sigma Aldrich (St. Louis, Catalysts 2021, 11, 1192 11 of 15 MO, USA) prior to use. The biocatalysts were also dried, albeit by placing the amounts corresponding to 10,000 AU in a dryer containing silica gel. Operational stability tests were performed by removing used reaction medium and rinsing the biocatalysts with heptane twice. Then, immobilized derivatives were allowed to stand in the dryer for 24 h before the next reaction run [44]. Initial esterification rates were determined by withdrawing samples regularly over the first 90 min of reaction. The relative yields of consecutive transesterification and esterification cycles as calcu- lated relative to the final yield of the first reaction cycle were fitted by using a first-order exponential decay model (1) and two-component first-order exponential decay model (2): Y(%)t = 100e−kt , (1) Y(%)t = 100e−k 1 t + ce−k 2 t (2) where k, k1 and k2 are deactivation coefficients. All computations were done with the software Sigma Plot v. 14 [52,65]. 3.9. Gas Chromatography Fatty acid methyl and ethyl esters (FAME and FAEE, respectively), and ethyl butyrate concentrations formed in the transesterification and esterification reactions, were deter- mined on a 7890A gas chromatograph from Agilent (Santa Clara, CA, USA) equipped with a 19095 N-123 capillary column and an autosampler [44,66]. The relative standard deviations (RSD) never exceeded 3%. 3.10. Immobilized Lipase Stability against Organic Solvents The stability of EB-proROL and EB-rROL against organic solvents was assessed by using closed vials of 10 mL. Thus, a total amount of 5000 AU of each biocatalyst was incubated with 5 mL of the corresponding solvent at 25 ◦ C under stirring at 350 rpm in the IKA KS 400 incubator for 24 h. Then, the biocatalysts were washed with heptane twice and the initial rate of ethyl butyrate production was determined as described in Section 3.8. All data given are relative to the initial reaction rate of the same amount of non-incubated biocatalyst for each lipase. 4. Conclusions The truncated prosequence of Rhizopus oryzae lipase was confirmed to suffice in order to alleviate the adverse effects on proROL and enable its expression in K. phaffii under the constitutive promoter PGAP with glucose and glycerol as substrates and hence to avoid the need for methanol. A more environmentally friendly bioprocess for proROL production afforded final activity and productivity values similar to those obtained with the best existing methanol feeding strategy. Preferential hydrolysis of the 28 amino acids in the truncated prosequence was demonstrated by N-terminal analysis (that is, the 28 amino acids were confirmed to be naturally designed for removal). In addition, the truncated prosequence increased the tolerance of organic solvents, and the operational stability in biodiesel and ethyl butyrate production, of EB-proROL relative to EB-rROL. However, no influence on initial reaction rates was observed, which suggests that EB-proROL and EB-rROL interact identically with the substrates under the studied conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/catal11101192/s1, Figure S1: rROL PAOX1 -plasmid: plasmid containing the sequence of rROL under PAOX1 . Figure S2: proROL PAOX1 -plasmid: plasmid containing the sequence of proROL under PAOX1 . Figure S3: rROL PGAP -plasmid: plasmid containing the sequence of rROL under PGAP . Figure S4: proROL PGAP -plasmid: plasmid containing the sequence of proROL under PGAP . Author Contributions: Conceptualization, J.L.-F., M.D.B. and F.V.; methodology, J.L.-F.; validation, J.L.-F., M.D.B. and F.V.; formal analysis, J.L.-F.; investigation, J.L.-F.; resources, M.D.B. and F.V.; data curation, J.L.-F.; writing—original draft preparation, J.L.-F.; writing—review and editing, M.D.B. Catalysts 2021, 11, 1192 12 of 15 and F.V.; visualization, J.L.-F.; supervision, M.D.B. and F.V.; project administration, M.D.B. and F.V.; funding acquisition, F.V. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Spanish Ministry of Science and Innovation (Project PID2019- 104666GB-100). Acknowledgments: JLF was supported by a Basque Government scholarship for the training of pre-doctoral researchers (PRE_2017_1_0110). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ROL Rhizopus oryzae lipase. rROL Recombinant Rhizopus oryzae lipase formed by the mature sequence. proROL Lipase formed by the C-terminal 28 amino acids of the native prosequence of ROL fused to the N-terminal in the mature sequence. proROLm proROL modified by the activity of proteases. w-proROL-gene: Gene encoding the whole prosequece fused to the mature sequence of Rhizopus oryzae lipase. rROL-gene Gene encoding the mature sequence of Rhizopus oryzae lipase. proROL-gene Gene encoding the C-terminal 28 amino acids of the native prosequence of ROL fused to the N-terminal of the mature sequence. EB Purolite® polymethacrylate matrix support with butyl and epoxide surface groups EB-rROL rROL covalently immobilized onto the EB support. EB-proROL proROL covalently immobilized onto the EB support. rROL PAOX1 -plasmid Plasmid containing the mature sequence of Rhizopus oryzae lipase under PAOX1 . proROL PAOX1 -plasmid Plasmid containing the sequence of proROL under PAOX1 . rROL PGAP -plasmid Plasmid containing the mature sequence of Rhizopus oryzae lipase under PGAP . proROL PGAP -plasmid Plasmid containing the proROL sequence under PGAP . rROL PAOX1 -strain Genetically modified Komagataella phaffii strain used to produce rROL under PAOX1 . proROL PAOX1 -strain Genetically modified Komagataella phaffii strain used to produce proROL under PAOX1 . rROL PGAP -strain Genetically modified Komagataella phaffii strain used to produce rROL under PGAP . proROL PGAP -strain Genetically modified Komagataella phaffii strain used to produce proROL under PGAP . MNLFB Methanol non-limited fed-batch. MLFB Methanol limited fed-batch. Y(P/X) Product biomass yield (AU gX −1 ). µ Specific growth rate (h−1 ). qp Specific production rate (AU gX −1 h−1 ): parameter which shows an average of all the analyzed points during the bioprocess. Specific productivity (AU gX −1 h−1 ) Parameter of the entire bioprocess, analyzed at the end of it. Volumetric productivity (AU L−1 h−1 ) Parameter of the entire bioprocess, analyzed at the end of it. PAOX1 Inducible alcohol oxidase 1 promoter. PGAP Constitutive glyceraldehyde-3-phosphate dehydrogenase Catalysts 2021, 11, 1192 13 of 15 References 1. Bornscheuer, U.T. The fourth wave of biocatalysis is approaching. Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 2018, 376, 20170063. [CrossRef] [PubMed] 2. Bilal, M.; Iqbal, H.M.N. Armoring bio-catalysis via structural and functional coordination between nanostructured materials and lipases for tailored applications. Int. J. Biol. Macromol. 2021, 166, 818–838. [CrossRef] [PubMed] 3. 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