Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate

Abstract A novel Lip+ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip+Amy+host. Lipase hydrolyzed methyloleate into methanol that sustained P AOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip+Amy+ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy+ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip+ host to validate disparity between lipase background and P AOX1 induction in presence of methyloleate.


Introduction
Pichia pastoris (also classified as Komagataella phaffii) expression system is widely used to produce various intra and extracellular proteins. [1][2][3] The system offers ease of genetic manipulations and being a eukaryotic system, it allows various post-translational modifications. Therefore, many proteins that are rendered inactive as inclusion bodies in Escherichia coli, can be produced as biologically active entities in P. pastoris. [2] Among other promoters under which foreign proteins can be easily cloned in this system, alcohol oxidase promoter (P AOX1 ) is widely preferred to attain high product yields. [4] The methanol inducible P AOX1 regulates the expression of enzyme alcohol oxidase, which catalyzes the first step of the methanol utilization pathway and constitutes 30% of total soluble protein when P. pastoris is grown solely on methanol, showcasing its prominent strength. [5] This tightly regulated promoter has been developed to uniquely govern the expression of heterologous proteins [6,7] and its strong inducibility in presence of methanol drives the expression of genes under its regulation. [4,8,9] However, the promoter poses limitations such as repression in the presence of carbon sources such as dextrose, ethanol, glycerol; [10] and for maintaining it in an induced state, multiple methanol inductions are required. Methanol is potentially hazardous due to its toxic and flammable nature, and storing large volumes poses risk. Further, methanol catabolism requires an enormous amount of oxygen, [11] and generates immense heat. [5] Supplementing oxygen and heat removal further increases the overall production cost, [12] and repetitive usage increases the contamination risks. [13] However, continually growing demand and market expansion for recombinant proteins mandate improvement in the protein production platforms, and current study provides an alternative to methanol induction in P. pastoris expression system, circumventing its drawbacks while retaining attributes like strength and inducibility of P AOX1 .
Earlier, Kumari and Gupta [14] reported expression of lipase from Y. lipolytica under P AOX1 using methyloleate as an alternate source of methanol induction. Here, P AOX1 induction was primarily initiated using methanol to express lipase and once sufficient levels were attained, methyl esters were supplemented in the production broth. Successively, methyl esters were hydrolyzed by lipase, and methanol released in the process initiated further P AOX1 induction. In this light, it was hypothesized that if Pichia host is made Lipase positive (Lip þ ) by expression of lipase gene under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, then a platform could be built where methyl esters such as methyloleate can be used as an alternate source of P AOX1 induction. In the present study, a system has been developed where a Lip þ host constitutively expressing lipase has been tested for expression of two reporter proteins, amylase (Lip þ Amy þ ) from Bacillus licheniformis and c-CGTase (Lip þ CGT þ ) from Evansella caseinilytica, under P AOX1 using methyloleate. Lipase produced in the culture broth hydrolyzed ester bond in methyloleate releasing methanol and oleic acid. Consecutively, methanol resulted in P AOX1 induction and oleic acid was utilized as a carbon source for biomass production while acting as an anti-foaming agent. Conclusively, in this study, we report an expression platform for sustained P AOX1 induction aiming to overcome drawbacks of repetitive methanol usage as an inducer by allowing the continual release of methanol from methyloleate.

Culture and reagents
Cultures viz wild-type strain P. pastoris X-33 was procured from Invitrogen (Waltham, Massachusetts, USA) and Y. lipolytica MSR 80 (MTCC number 9517) was obtained from the laboratory culture collection. Restriction enzymes, Taq Polymerase, and T4 DNA Ligase were procured from New England Biolabs (NEB, Ipswich, Massachusetts, USA); antibiotics kanamycin and geneticin were purchased from Himedia (Mumbai, Maharashtra, India), and zeocin from Invitrogen (Waltham, Massachusetts, USA). GSure Plasmid Isolation Minikit, was procured from GCC Biotech (Joychandipur, West Bengal, India) and DNA gel extraction kit was purchased from Qiagen (Hilde, NRW, Germany); fungal DNA isolation kit was purchased from Zymo Research (Irvine, California, USA). Luria Bertani (LB) was purchased from Difco TM (Franklin Lakes, New Jersey, USA) and other culture media components viz Yeast Extract, Peptone, Dextrose, Yeast Nitrogen Base were purchased locally from Himedia. Methanol was procured from Merck (Darmstadt, Hesse, Germany) while biotin, methyloleate, pNp esters and 3, 5-Dinitrosalicylic acid (DNSA) were purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade.

Construction of lipase positive (lip 1 ) host
The sequence of lipase gene lip2 of Y. lipolytica MSR80 was acquired from NCBI (JQ396255.1) and was found to be intron free. The gene (was amplified from the genomic DNA of Y. lipolytica MSR80 using primers Lip2FP and Lip2RP (Table 1). The amplified gene was digested with EcoR1 and Kpn1 and ligated into the eukaryotic shuttle vector pGAPZaA. The construct pGAPZaAlip2 was then transformed into E. coli DH5a competent cells. The recombinants were screened on low salt (0.5% NaCl) LB (pH 7.5) plates supplemented with zeocin at a concentration of 25 mg/ml. Colony PCR was performed to initially screen the recombinants and plasmid was isolated from positive clones. Restriction digestion was performed to determine the size of the fall-out and the recombinant plasmid was further sequenced using lip2 specific forward and reverse primers to confirm the gene sequence (Supplementary Figure 1A). The positive recombinant DH5a culture was grown overnight at 37 C/200 rpm in the presence of 25 mg/ml zeocin and recombinant plasmid pGAPZaAlip2 was isolated. It was further linearized with AvrII and transformed by electroporation into competent P. pastoris X33 prepared by the lithium acetate method. [15] The transformation was performed by electroporation using BIORAD MicroPulser at Pichia settings in 0.2 cm Gene Pulser cuvettes. 5 mg of linearized pGAPZaAlip2 in the total volume of 20 ml was used for transformation. 100 ml of the transformation mix was then spread platted onto YPD sorbitol plates supplemented with zeocin at a concentration of 200 mg/ml to select the recombinant clones. The plates were incubated at 30 C for 48 h and the colonies obtained here were further screened for lipase production.
Recombinant Lip þ clones were initially screened on YPD (0.5% w/v Yeast Extract, 1% w/v Peptone, 1% w/v Dextrose) agar plates containing 1% tributyrin and rhodamine B plates containing 1% olive oil. [16,17] The plates were incubated at 30 C for 48 h and colonies showing zone of hydrolysis (Supplementary Figure 1B,C) on these plates were selected further in the study for developing an alternate expression system to avoid methanol usage.
Alternatively, another lipase Lip11 was utilized in developing a Lip þ host to show P AOX1 induction in presence of methyloleate irrespective of lipase. The sequence of lip11 was procured from NCBI (>JQ396256.1) and it was amplified from the genomic DNA of Y. lipolytica MSR80 using gene-specific primers ( Table 1). The amplified product was cloned into pGAPZaA and the clones were initially screened by colony PCR and confirmed by fall-out analysis using EcoR1 and Kpn1 restriction enzymes (Supplementary Figure  S2) followed by DNA sequencing. The positive plasmids were linearized with AvrII and transformed into competent P. pastoris X33 cells and Lip þ clones were screened as described earlier.

Growth of lip 1 host and expression of Lip2
A single colony of P. pastoris X33 and Lip þ host was inoculated into YPD broth and incubated for 24 h at 30 C, Table 1. Primer sequences were used for gene amplification in the study.

Primer Sequence
Lip2FP: rpm. This was used as primary culture and inoculated at 2% into fresh YPD media, incubated at 30 C, 250 rpm and OD 600 was measured at different time intervals. Lipase activity was also assayed at different time intervals using pNppalmitate. Wild-type P. pastoris X33 was used as a control.

Lipase assay
Lipase assay was performed using p-nitrophenyl palmitate (C16). [18] Briefly, 800 ml of 1 mM p-nitrophenyl palmitate (C16) was mixed with 200 ml of cell-free culture broth and incubated at 50 C for 10 min. Following incubation, 20 ml of Triton X-100 was added to remove turbidity and absorbance was measured at 410 nm. One lipase unit was defined as the amount of enzyme required to release 1 mmole pnitrophenol per min at optimum pH and temperature.
Cloning and expression of amylase in Lip 1 Amy 1 host The sequence of amylase gene Amy of B. licheniformis was acquired from NCBI (NC_006270.3: 657584-659122). The gene was then amplified from the genomic DNA of B. licheniformis ER15 without its native signal sequence using primers AmyFP and AmyRP ( Table 1). The amplified gene was digested with EcoR1 and Xba1 and ligated into the pPICKaA vector, where zeocin resistance cassette from pPICZaA vector is replaced with kanamycin resistance cassette. [19] The construct pPICKaAamy was then transformed into E.coli DH5a competent cells. The recombinants were screened on LB plates supplemented with kanamycin at a concentration of 50 mg/ml. The recombinant plasmids were confirmed by fall-out analysis (Supplementary Figure 3A) and DNA sequencing. The positive plasmid was then linearized with SacI and transformed by electroporation into a competent P. pastoris X33 host and Lip þ host, prepared by the lithium acetate method as mentioned earlier. The recombinants were screened and selected on YPD agar plates containing geneticin at a concentration of 250 mg/ml. Recombinant clones were screened for amylase production in buffered media up to 48 h (induction with 0.5% methanol every 12 h), following which they were qualitatively assayed on 1% starch agar plate using 200 ml of cell-free culture broth (Supplementary Figure 3B,C). [19,20] Expression of amylase in Lip 1 Amy 1 host and westernblot analysis Amylase was expressed in recombinant Lip þ Amy þ host as per Invitrogen protocol in buffered media (0.5% Yeast Extract, 1% Peptone, Yeast Nitrogen Base, pH 6.5) and induction was done using 0.5% methyloleate, every 24 h. Briefly, a single recombinant clone was inoculated in 10 ml YPD broth and incubated at 30 C, 200 rpm, 24 h and was used as primary inoculum to inoculate BMGY (buffered media containing 1% glycerol) at a final OD 600 of 0.1. The culture pellet was harvested after it reached desired OD 600 and resuspended into BMMY (buffered methanol-complex medium) containing methyloleate to induce amylase expression. The P AOX1 induction was maintained by supplementing 0.5% (v/v) methyloleate every 24 h. The Amy þ host harboring amylase expression cassette under P AOX1 was used as comparative control wherein P AOX1 was maintained in an induced state by supplementing 0.5% (v/v) methanol every 12 h. Samples were withdrawn at regular time intervals postinduction and amylase production was quantified. Further, expression was analyzed on 12% SDS-PAGE followed by western-blot analysis using monoclonal Anti-His Tag (HRP Conjugated) antibody (Chongqing Biospes Co Ltd, Chongqing, Chongqing Shi, China). The blots were developed using diaminobenzidine and hydrogen peroxide. Amylase expression was presented in terms of both volumetric yield (amylase units present in 1 ml of extracellular production broth) and yield (amylase titers produced per milligram dry cell weight).

Amylase assay
Amylase was quantified using 1% starch prepared in phosphate buffer pH 7 employing DNSA (3, 5-Dinitrosalicylic acid) method. [21] The reaction was set using 0.5 ml of appropriately diluted culture broth and 0.5 ml 1% starch solution and incubated at 50 C, 150 rpm for 10 min. 1 ml DNSA was then added and samples were boiled at 100 C for 10 min.
The samples were diluted with 18 ml distilled water and absorbance was measured at 540 nm. One amylase unit was defined as the amount of enzyme required to release 1 mmole glucose per minute at optimum pH and temperature.

Oleic acid utilization
Oleic acid utilization was studied using mixed feed culture broth (BMMY) containing both methanol and oleic acid. Oleic acid was used at a concentration of 0.5% and 1% methanol was supplemented every 12 h. Culture broth supplemented only with 0.5% oleic acid was used as a control. Sample aliquots were withdrawn at different time intervals and oleic acid was detected using Gas Chromatography. 500 ml sample was acidified with 5 ml concentrated hydrochloric acid and mixed with an equal volume of hexane for oleic acid extraction. The top hexane layer was carefully removed and 10 ml of this sample was analyzed on Gas Chromatography. Percent oleic acid utilization was calculated as percent reduction in the peak area of oleic acid at different time intervals, using peak area obtained at 0 h as 100%.
Detection of methyl oleate and oleic acid using gas chromatography The concentration of methyl oleate and oleic acid was monitored by Gas Chromatography using the following conditions in Stabil Wax Another reporter protein c-CGTase from E. caseinilytica was used to validate the functionality of the developed platform to express heterologous proteins under P AOX1 using methyloleate. Recombinant clone expressing c-CGTase (CGT þ ) under the control of P AOX1 was available in the lab and was used in developing the platform. Lip2 was cloned into vector pGAPKaA (consisting of a kanamycin resistance cassette, developed earlier in the lab) using gene-specific primers ( Table 1). The recombinant vector was then transformed into a competent CGT þ host and the positive clones (Lip þ CGT þ ) were screened and selected using YPD-Tributyrin plates as described earlier. The protein was purified from the cell-free production broth by affinity chromatography using Ni 2þ -NTA Superflow resin (Qiagen, Germany) and eluted in 50 mM imidazole (20 mM phosphate buffer pH 7).
c-CGTase assay c-CGTase activity was quantified as per the standard protocol described earlier by Takada et al. [22] The reaction was set using 0.5 ml of the appropriately diluted enzyme along with 0.5 ml of 1.5% potato starch prepared in 50 mM Glycine-NaOH buffer pH 11. The reaction was incubated at 50 C for 20 min following which it was terminated by adding 100 ml of 5 mM bromocresol green dye prepared in 20% ethanol. The reaction was further incubated for 20 min at 28 C and 2 ml of 1 M acetate buffer (pH 4.2) containing 30 mM citric acid was added before measuring absorbance at 630 nm. c-CGTase activity was expressed as mmoles of c-cyclodextrin synthesized and it was calculated using the regression equation obtained using standard c-cyclodextrin (y ¼ 0.3309x for c-CD). One enzyme unit was defined as the amount of enzyme required in producing 1mmole of c-cyclodextrin per min under standard assay conditions.

Results and discussion
Growth and expression of Lip 1 host Lip þ host was generated by cloning lip2 from Y. lipolytica under the constitutive GAP promoter of wild-type host P. pastoris X33 by genomic integration of the expression construct. The growth of the Lip þ host was monitored along with the wild-type host in YPD media. From the growth profile, it was observed that both Lip þ and wild-type hosts exhibited similar growth patterns (Supplementary Figure 4). Lipase activity in the culture broth of Lip þ host increased with incubation time ( Table 2) and its production did not alter the growth pattern. Therefore, the Lip þ host was taken further for expression of reporter protein amylase under P AOX1 using methyloleate.

Oleic acid utilization by Pichia host in presence of methanol
The lipase in the Lip þ host was expressed under the constitutive GAP promoter as an extracellular secretory protein by virtue of an a-secretory signal peptide at its N-terminus. [23] Therefore, lipase present in the extracellular culture broth of Lip þ host would result in hydrolysis of methyloleate into methanol and oleic acid, later of which is utilized by the host as a carbon source. However, sustained release of methanol could inhibit oleic acid utilization due to its catabolite repression [24] in the presence of methanol. It was crucial to confirm oleic acid utilization by the P. pastoris host in presence of methanol before examining the expression system. Therefore, wild-type X33 host was subjected to mixed-fed cultivation to study catabolite repression of oleic acid utilization in the presence of methanol. A methanol concentration of 1% (supplemented every 12 h) was selected because methanol released from methyloleate hydrolysis would never reach this high concentration, and moreover, higher concentrations of methanol are toxic to cellular viability. [2] Additionally, in a previous study, high methanol concentration (2%) was shown to be repressive for oleic acid utilization. [14] The fermentation broth from a mixed-fed batch containing both oleic acid and methanol was analyzed on Gas Chromatography at different time points (Figure 1). In both, the presence and absence of methanol, similar percent oleic acid utilization was observed at 6 h and 12 h indicating that oleic acid utilization was not catabolically repressed in the presence of 1% methanol. However, in mixed-fed cultivation, complete oleic acid utilization required 48 h while only 24 h in the absence of methanol, possibly because co-utilization of two carbon sources delayed complete oleic acid utilization. Earlier studies have also reported induction of a family of peroxisomal enzymes, particularly, fatty acid b-oxidation pathway enzymes [25,26] when P. pastoris was cultivated on oleic acid. In the current study, oleic acid was efficiently utilized as a carbon source by the Pichia host even in presence of 1% methanol. Therefore, it could be clearly substantiated that this concentration of methanol does not affect oleic acid utilization, and in the present system, methanol concentration would never exceed this level.
System validation: expression of a-amylase in Lip 1 Amy 1 host Amylase expression was successfully obtained in the Lip þ Amy þ host using methyloleate as an alternate source of methanol induction. Amylase expression was confirmed both quantitatively using DNSA and by SDS-PAGE analysis. Upon SDS-PAGE analysis, amylase was observed in the extracellular culture broth as approximately 70 kDa band which was further confirmed by western blot analysis using an anti-His antibody (Figure 2). The initial system validation of P AOX1 induction using methyloleate was done at induction OD 600 of 4. In presence of methyloleate, Lip þ Amy þ host resulted in total amylase production of 1927.7 ± 30 Â 10 3 U/L with a yield of 175.2 ± 2 U/mg biomass at 48 h. Amylase production levels achieved in Lip þ Amy þ host were comparable to Amy þ host, where upon induction with 0.5% methanol every 12 h, total amylase production of 1906.7 ± 6Â10 3 U/L with a yield of 161.6 ± 0.5 U/mg biomass was obtained. Therefore, the Lip þ Amy þ host successfully led P AOX1 induction in the presence of methyloleate by virtue of constitutively expressed lipase in this host. Earlier studies have reported around 1600 U/ml of this thermostable amylase from B. licheniformis in P. pastoris expression system using methanol inducible P AOX1.
[27] The current study revealed comparable results where methyloleate was utilized successfully instead of methanol in a Lip þ Amy þ host. Further, lipase levels were sufficient to catalyze the hydrolysis of methyloleate, allowing the sustained release of methanol and maintaining P AOX1 in an induced state. Furthermore, comparable amylase levels in Lip þ Amy þ and Amy þ host suggested that lipase production in Lip þ Amy þ host did not affect amylase expression by exerting an additional burden on the cellular protein machinery. Methyloleate hydrolysis in the culture supernatant of Lip þ Amy þ host was also monitored by Gas Chromatography (Figure 3) where an apparent reduction in the peak area of methyloleate was observed with the release of oleic acid.

Methyloleate concentration for optimal P AOX1 induction
The concentration of methyloleate for expression of heterologous protein in Lip þ Amy þ host was optimized in a range of 0.5%, 1%, 1.5% and 2% methyloleate, supplemented into production medium every 24 h. A concentration of 0.5% methyloleate resulted in total amylase production of 2237.8 ± 55 Â 10 3 U/L yielding 186.5 ± 4.6 U/mg biomass at 48 h ( Figure 4). However, supplementing higher concentrations of methyloleate resulted in no further increase in volumetric yield or biomass generation. Therefore, methyloleate concentration of 0.5% was found ample enough in substantiating P AOX1 induction and oleic acid released was sufficiently exhausted by the Lip þ Amy þ host in generating productive biomass.

System validation at higher induction OD 600
The developed system functioned as anticipated and the presence of methyloleate successfully resulted in P AOX1 induction to drive amylase expression. In order to further check sustainability of the system, expression studies were conducted for prolonged intervals using higher induction biomass. An induction OD 600 of 8-10 was used, and amylase production was studied up to 196 h using 0.5% methyloleate supplemented every 24 h as a source of methanol to maintain the induction level of P AOX1 ( Figure 5). Under these conditions, Lip þ Amy þ host produced maximum amylase units of 4268.0 ± 232 Â 10 3 U/L at 120 h; accordingly resulting in a yield of 221.0 ± 10 U/mg biomass. The production levels achieved were equivalent to Amy þ host where maximum amylase units of 4002.6 ± 4Â10 3 U/L were obtained at 132 h resulting in 203.6 ± 4 U/mg biomass. Although the production levels obtained with Lip þ Amy þ host were comparable to the Amy þ host, maximum yield in the case of Lip þ Amy þ host could be achieved at earlier hours (120 h in Lip þ host vs 132 h in wild-type host). The samples from different time intervals were also analyzed on SDS-PAGE and protein was observed as a distinct band at 70 kDa size ( Figure 6).
Upon further increasing the induction OD 600 to 18-20, Lip þ Amy þ host produced maximum amylase units of 4776.0 ± 45 Â 10 3 U/L at 96 h resulting in a yield of 220.9 ± 13 U/mg biomass (Figure 7). The levels achieved in Lip þ Amy þ host were again comparable to the Amy þ host where maximum amylase production of 4382.7 ± 189 Â 10 3 U/L was obtained at 108 h when P AOX1 was maintained in an induced state by supplementing 0.5% methanol every 12 h. Maximum yield was also achieved at 108 h with a value of 206.0 ± 17 U/mg biomass. The cell-free supernatants withdrawn at different time intervals were also analyzed on SDS-PAGE, where amylase was observed as a distinct 70 kDa band ( Figure 8). Further, reduction in the amylase levels observed after certain time periods during high cell-density cultivation could be due to preolytic degradation of the expressed protein. Intracellular proteases are released into the extracellular milieu due to disrupted cell membranes at high cell densities. [28] These proteases result in hydrolysis of the extracellularly expressed protein and hence a decrease in levels is observed after certain time intervals during prolonged cultivation periods.
Amylase production at higher induction OD 600 ¼ 8 or 20 accordingly showed that Lip þ Amy þ host produced increased biomass in the presence of methyloleate as compared to Amy þ host induced using methanol. Further, at both induction OD 600 of 8 or 20, maximum production was obtained at earlier hours in Lip þ Amy þ host using methyloleate. Oleic acid released during methyloleate hydrolysis emulsified well in the production broth and utilized efficiently as a carbon source. Similar values were achieved in Amy þ host at later hours, where induction was done with methanol. Further, as improved yields were attained using Lip þ Amy þ host in the presence of methyloleate, it could conclusively be implied that it was the sustained productive biomass leading to enhanced volumetric yields.
Wang et al. [29] earlier reported the heterologous expression of alpha-amylase from B. licheniformis in P. pastoris. A total of 3500 U/ml of alpha-amylase was obtained upon methanol induction in a 5-L fed-batch bioreactor which increased to 8100 U/ml upon codon optimization. However, comparable production levels (non-optimized) were achieved in the present work even at a shake-flask level using methyloleate as an alternate source of methanol induction in Lip þ Amy þ host.

Platform validation using alternate lipase Lip11
Alternate lipase Lip11 was utilized in developing a Lip þ host to validate the functionality of methyloleate in inducing P AOX1 irrespective of lipase employed. The lipase activity in the production broth as a function of time has been presented in Supplementary Table 1. As expected, irrespective of lipase utilized in developing a Lip þ host, maximum amylase production was attained at earlier hours in Lip þ Amy þ host by incorporating 0.5% methyloleate into the production broth every 24 h. A maximum of 4067.1 ± 77 Â 10 3 U/L was attained at 108 h in Lip þ Amy þ host resulting in a yield of 234.6 ± 8 U/mg biomass (Figure 9). While Amy þ host produced 3954.2 ± 73 Â 10 3 U/L at 120 h with a yield of 222.4 ± 7 U/mg biomass (Figure 9) upon induction with 0.5% methanol every 12 h. The cell-free supernatants were also analyzed by SDS-PAGE where amylase could be visualized as a sharp band of approx. 70 kDa (Supplementary Figure 5).
These observations were in coherence with earlier results where Lip2 was utilized in developing a Lip þ host.
Reproducible results indicate that a Lip þ host can serve as a platform for the expression of heterologous proteins under P AOX1 irrespective of lipase utilized in developing the host. Methyloleate can successfully be utilized by this host resulting in P AOX1 induction while serving as a carbon source for biomass production.
Platform validation with second reporter protein c-CGTase c-Cyclodextrin glycosyltransferase (c-CGTase) catalyzes the synthesis of c-cyclodextrins using various starch substrates, and the product c-cyclodextrins find wide applicability in food and pharmaceutical industries. [30] c-CGTase from E. caseinilytica was employed as the second reporter protein to show the applicability of the developed platform in  expressing various heterologous proteins using alternate methanol sources such as methyloleate.
Total c-CGTase production of 49.8 U/L ± 1.7 was achieved in a CGT þ host at 72 h upon induction with 0.5% methanol every 12 h (Figure 10). On the proposed platform (Lip þ CGT þ ) however, 51.5 U/L ± 1.4 were attained in the presence of 0.5% methyloleate (supplemented every 24 h). The purified c-CGTase could be visualized as a distinct band of approx. 75 kDa ( Figure 10) on a 12% SDS-PAGE gel. The results clearly reflect the applicability of the proposed platform for the expression of heterologous proteins in presence of alternate methanol sources such as methyloleate.
The reported platform is a versatile system that can be easily generated by constitutive expression of any available lipase capable of hydrolyzing methyl esters. As a proof of concept, comparable productivity of heterologous amylase and c-CGTase was achieved with the developed host as obtained with the classical host, induced with repetitive methanol usage. The expression platform functioned efficiently even at higher cell densities, and the time required to achieve maximum production levels was reduced. The developed system overcomes the drawbacks of widely used methanol such as toxicity along with risks of hazard and contamination due to repetitive usage and shall serve well as a novel platform for expressing various heterologous proteins using methyl esters as effective methanol substitutes for P AOX1 induction.