Cloning and Expression of an Active Aspartic Proteinase Gene From Aspergillus Oryzae DRDFS13 in Pichia Pastoris

Background Pichia pastoris is


Background
Acid proteases are widely utilized in the food, beverage, and pharmaceutical industries.The major application of acid proteases is in cheese production in the dairy industry.Several species of fungi such as Aspergillus, Candida, Rhizomucor and Phanerochaete chrysosporium are reported to yield high amounts of aspartic protease enzymes while Aspergillus spp is the major producers for they possess several encoding genes such as pepA (Gomi et al., 1993;Nair & Jayachandran, 2019).
Aspartic protease genes from fungi may be expressed in yeast for large scale fermentation as it is reported that yeast is good expression hosts for genes of fungal origin (Sun et al., 2018;Yegin and Fernandez-Lahore, 2013).The authors reported that the methylotrophic yeasts such as Pichia pastoris are widely used as expression platforms for recombinant proteins for basic research and industrial applications.
The methylotrophic yeast Pichia pastoris, currently reclassi ed as Komagataella pastoris, has become a substantial workhorse for biotechnology, especially for heterologous protein production (Ahmad et al., 2014).P. pastoris have many advantages in yielding a high-level expression of recombinant proteins, protein processing and are characterized by post-translational modi cations (Kangwa et al., 2018;Luo et al., 2016).The post-translation modi cation associated with higher eukaryotes such as processing of signal sequence, folding, disul de bridge formation, certain types of lipid addition and Oand N-linked glycosylation (Cereghino and Cregg 2000).It can also be cultivated on inexpensive media with low-level proteins and has been accepted as a safe and effective expression system by the U.S. Food and Drug Administration (FDA) (Luo et al., 2016).P. pastoris has been shown great achievement in the large-scale production of recombinant protein (Cregg et al. 2000).
The expression of the aspartic protease in P. pastoris is achieved by cloning the protease gene into the expression vector pGAPZα-A.pGAPZαA is chosen as an expression vector since it is designated for highlevel constitutive expression in P. pastoris (Cereghino and Cregg 2000).The pGAPZαA was created when the methanol-regulated AOX1 promoter was replaced with a constitutive, glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in the vector.The advantage of using the GAP promoter is that there is no need to shift cultures from one carbon source to another as methanol is not required for induction of the enzyme.It is established that genetic manipulation in lamentous fungi is more complex than in yeast and bacteria and so necessitates expression of the desired gene from lamentous fungi into a suitable host.For the last two years, we collected and screened several bacteria and fungi for their aspartic protease activities in our laboratory, of which, some of the local lamentous fungi showed potential for aspartic protease activity in a pilot-scale study.
The aim of the present work is to study the expression of an aspartic protease gene from the local A. oryzae DRDFS13 strain into P. pastoris.The gene was cloned in pGAPZαA and inserted into E. coli K-12 for plasmid ampli cation and transformed in P. pastoris for protein expression.The milk-clotting activity of the protein was investigated for its potential in cheese manufacturing.

Fungal and bacterial strain
The microorganism used as the source of the gene encoding aspartic protease enzyme was Aspergillus oryzae DRDFS13.The fungal strain was grown at 30 0 C for 3 days in a liquid broth (Potato Dextrose Broth).E. coli K-12 ER2738 was used to amplify the plasmids carrying the cloned gene.E. coli strains were grown overnight in Luria-Bertani medium (10 g L -1 , tryptone, 5 g L -1 yeast extract, 5 g L -1 NaCl) at 37 °C, 220 rpm.P. pastoris X-33 was grown in YPD medium (10 g L -1 yeast extract, 20 g L -1 peptone, 20 g L -1 glucose) at 30 °C for 3 days with shaking at 250 rpm (Kangwa et al. 2018).

cDNA synthesis
First, total RNAs were extracted from the mycelia of Aspergillus oryzae DRDFS13 using a NucleoSpin® RNA extraction kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's standard protocol (Yegin and Fernandez-Lahore, 2013).Then, the rst-strand cDNA was synthesized from RNA using the ProtoScript® II First Strand cDNA Synthesis Kit (#E6560S, New England Bio Labs Inc, Brüningstraße 50, 65929 Frankfurt am Main) according to the manufacturer's protocol.The reaction components were mixed and incubated at 42 o C for 1 h, followed by heat inactivation at 80 o C for 5 min.Products were then stored at -20 o C for further ampli cation of the aspartic protease gene (Antonio et al., 2013).
2.3.Aspartic protease gene ampli cation and sequencingAmpli cation of the aspartic protease gene was done using forward primers APJM_Fw01 5'-CCT CGA GCA TGG TTA TCT TGA GCA AAG TCG C-3' and reverse primer APJM_Rw01 5'-GCG GCC GCC AAG CCT GGG CGG CGA AGC CGA G-3', other PCR components were: 10 µl of 5x Phusion High Fidelity buffer, 1µl of 10 mM dNTPs solution mix, 0.5 µl of 2,000U Phusion High delity Polymerase, all purchased from New England Bio Labs Inc, Frankfurt am Main, 2.5 µl of 10 mM primer (forward and reverse) synthesized by Euro ns genomics, 2 µl of plasmid DNA (50 ng/µl), and sterile distilled water was added to the nal volume of 50 µl.From the ampli ed DNA, 15 µl of sample was run on a 1% agarose gel (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with 1X TPE buffer [stock concentration, 10X: 1 M Tris base, 20 mM EDTA, 225 mM phosphoric acid all products of Applichem GmbH, Darmstadt, Germany] at 90 V, maximum Amps for 55 minutes.The gel was stained in ethidium bromide solution and viewed using a Gel documentation system.The remaining PCR product (gene) was puri ed using a Nucleospin Plasmid Kit according to the manufacturer's protocol as described by Macherey-Nagel (2012).The resulting samples were eluted in 30 µl of elution buffer; concentrations were measured using a Nanodrop-2000 Spectrophotometer.Samples were then sent to Euro ns Genomics for sequencing.The results were analyzed in comparison to the protein amino acid sequence of Aspergillus oryzae RIB140 Aspartic protease sequences (Antonio et al., 2013).
2.4.Cloning and expression of aspartic proteinase gene in pastorisThe aspartic protease gene was inserted into pGAPZαA using the restriction enzymes XhoI and NotI (both from New England Bio Labs Inc) thereby producing an expression vector pMK-AP with the 6xHis tag at the C-terminal and kanamycin for selection in bacteria, and while zeocin as for selection in yeast (Yegin and Fernandez-Lahore, 2013).
Then the pMK-AP vector was transformed to E.coli K-12 ER2738 competent cells by electroporation at 1.8 kV for 5 milliseconds (Yegin and Fernandez-Lahore, 2013).
The tubes carrying competent cells were incubated at 37 0 C with shaking for 60 min.Then two types of samples (concentrated and un-concentrated) were prepared for plating.For the un-concentrated samples, 100 µl was taken from the Eppendorf tube directly after incubation.While the preparation of the concentrated samples involved centrifugation for a minute (11000 rpm at 4 0 C).Then 700 µl of the supernatant was discarded and the pellet was re-suspended in the remaining 200 µl.The cells were cultivated on LB agar plates supplemented with 25 μg/mL tetracycline and 25 μg/mL Kanamycin nal concentrations (Carl Roth GmbH, Karlsruhe, Germany) and incubated overnight at 37 0 C. Positive colonies carrying the coding sequence of aspartic protease gene were identi ed by colony PCR by lysing a colony in 10μL sterile water heated to 100 0 C for 10 minutes.One microliter of the lysed colony was further used in colony PCR using previously used primers and sequencing (Yegin and Fernandez-Lahore, 2013).
Plasmids carrying the AP coding sequence were extracted from positive colonies using a NucleoSpin® Plasmid Isolating Kit and further digested with AvrII restriction enzyme and inserted into P. pastoris X-33 using a heat shock method at 42 0 C for 2 min and the cells were screened on YPD agar plates containing 40 µg mL -1 zeocin.Transformants carrying the AP coding sequence was identi ed by PCR ampli cation using the previous primers and veri ed by nucleotide sequencing (Yegin and Fernandez-Lahore, 2013).

Cultivation
Colonies from P. Pastoris X-33 strain (control) and P. Pastoris X-33 aspartic protease (X-33 AP) were cultivated on YPD broth at pH 5 and pH 7 and incubated overnight at 30 0 C.For protein expression, 1 mL of X-33-AP was added into 3 asks containing 75 mL of YPD (pH 5) media and 1 mL of X-33 was added in 1 ask with 75 mL of YPD (pH 5) media.The same was done for YPD media with pH 7. The asks were incubated at 30 0 C for 6 days in a shaker incubator at 225 rpm.Samples were collected on 2 nd , 4 th and 6 th days and centrifuged at 4000 rpm and 4 o C for 30 min.Then, the supernatant was used as a crude enzyme (Yegin and Fernandez-Lahore, 2013).

Milk clotting activity
The milk-clotting activity of the enzyme was undertaken according to (Arima et al., 1970).Accordingly, 0.1 mL of the crude enzyme was added to 1 mL of reconstituted skim milk (Nestle TM) in 10 mL test tubes pre-incubated at 35 °C for 10 min.Reconstituted skim milk (NestleTM) solution consisted of 10 g dry skim milk/100 mL, 0.01 M CaCl 2 (AppliChemTM).The appearance of the rst clotting akes was visually evaluated and quanti ed in terms of Soxhlet units (SU).The endpoint was recorded when discrete particles were discernible.The clotting time T (s), the period of time starting from the addition of crude enzyme to the appearance of the rst clots and the clotting activity was calculated using the following formula: SU = (2400 * 5 * D) / (T * 0.5) Where T = clotting time (s) D= dilution of crude enzyme One SU is expressed as the quantity of enzyme required to clot 1 ml of a solution comprising 0.1 g skim milk powder and 0.01M calcium chlorides at 35 0 C within 40 min.

Protein determination
Protein was determined according to the Bradford procedure utilizing bovine serum albumin as the standard (Yegin and Fernandez-Lahore, 2013).

SDS-PAGE Analysis
When necessary, the crude enzyme extracted from recombinant P. pastoris was concentrated at room temperature using a Vacuum Concentrator 5305 (Eppendorf, Hamburg, Germany).Then, 40 μL of the samples (concentrated and non-concentrated) were loaded on a 12.5% SDS-polyacrylamide gel.For the molecular marker, 10 μL of Colour Protein Standard (New England Biolabs TM) was loaded.Electrophoresis was run for 50 min at 300 V and 60 mA.The gel was then stained with Coomassie Brilliant Blue (AppliChem TM) overnight and then detained overnight with the distaining solution (Kangwa et al., 2018).

Data analysis
Data analyses were performed using SAS software version 9 (Inc.Cary NC USA).The experiments were carried out in duplicate.Mean comparisons were done by Duncan's multiple range tests at the p-value of 0.05.
The puri ed gene of interest was later sent for sequencing to Euro ns Genomics with the primers used in the gene ampli cation.The sequence results were analyzed in comparison to the gene sequence of aspartic protease from Aspergillus oryzae RIB40.Accordingly, the amino acid sequence alignment of the aspartic protease gene from Aspergillus oryzae DRDFS 13 showed 98% similarity with the aspartic protease gene from A. oryzae RIB40 (Fig. 3.2).This con rmed the presence of catalytic Asp residues in the ampli ed gene from A. oryzae DRDFS13 (Antonio et al., 2013).Similarly, the deduced amino acid sequence for milk-clotting acid protease (MCAP) from M. circinelloides showed 88% similarity with M. bacilliformis PEP A gene (Antonio et al., 2013).The deduced amino acid sequence of the PEPA gene from A. oryzae has also shown 67% homology to the PEP A gene of A. awamori (Gomi et al., 1993).In another study, the gene encoding aspartic proteinase of M. mucedo had 80 % similarity with the Rhizopus niveus gene for aspartic proteinase II and 73 % similarity with rhizopuspepsinogen precursor of Rhizopus microsporus var.chinensis (Yegin and Fernandez-Lahore, 2013).

Cloning and expression of aspartic proteinase gene in pastoris
The concentrations of pGAPZαA vector (Fig. 3.3 and expression vector pMK-AP (Fig. 3.4) were determined by NanoDrop 2000 and found 186.4 ng/ μL 55.8 ng/ µL.The aspartic proteinase gene from Aspergillus oryzae DRDFS 13 was successfully expressed in P. pastoris under the control of the GAP promoter.Antonio et al., (2013), reported the expression of the aspartic protein gene from M. circinelloides in P. pastoris under the control of the constitutive GAP promoter.Similarly, the gene encoding an aspartic protease (MCAP) from M. circinelloides DSM 2183 cloned in P. pastoris was successfully expressed using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF) (Kangwa et al. 2018).
A novel aspartic protease gene (RmproA) from Rhizomucor miehei CAU432 cloned into P. pastoris was also successfully expressed in P. pastoris (Sun et al. 2018).In another study, an alkaline protease gene from A. oryzae and a serine protease gene from Thermoascus aurantiacus var.levisporus were cloned in P. pastoris GS115 were fruitfully expressed in P. pastoris (Guo & Ma, 2008;Li et al., 2011).

Milk clotting activity
The milk clotting activity of the crude extract collected from P. pastoris X-33 AP (transformed) and P. pastoris X-33 (wild type, control) was determined at day 2, 4 and 6 days.The highest MCA (190.47 U/mL) and speci c activity (30.75 U/mg) of the secreted crude enzyme from the recombinant yeast were obtained at pH 5 after 6 days of incubation (3.1).Similarly, the maximum MCA recorded for recombinant enzyme (210 U/mL) from M. mucedo DSM 809 expressed in P. pastoris (Yegin and Fernandez-Lahore, 2013) and the recombinant MCAP (257 CU/mL) from M. circinelloides expressed in P. pastoris (Antonio et al. 2013) was comparable with this study.However, the milk-clotting activity recorded from recombinant P. pastoris with plant milk-clotting aspartic protease (23 CU) (Feijoo-siota et al. 2018) and recombinant P. pastoris with bovine chymosin B (96 IMCU/mL) (Noseda et al. 2013) was lower than the present study.In another study, high protease activity (3480.4U/mL) was also recorded from an extracellular protease extracted from recombinant P. pastoris (Sun et al. 2018).
The data also showed substantial milk-clotting activity (MCA) of the recombinant yeast was obtained at pH 5; whereas, the crude enzyme from recombinant yeast cultivated on YPD media at pH 7 did not possess milk-clotting activity.This could be due to the fact that aspartic proteases may be produced under acidic conditions indicating that the initial pH of the media plays an important role in protein expression.Antonio et al., (2013) also reported the highest milk-clotting activity for aspartic protease from recombinant yeast (X-33/pGAPZα+MCAP-5) in YPD medium at pH 5.0.Interestingly, the enzyme was even induced in Pichia pastoris X-33 from M. mucedo DSM 809 at an initial medium pH of 3.5 (Yegin and Fernandez-Lahore, 2013).On the other hand, maximum enzyme activity was detected from recombinant P. pastoris at 72 h showing a difference in activity as a function of time (Antonio et al., 2013;Yegin and Fernandez-Lahore, 2013).The differences in the time of maximum milk-clotting activity production could be due to the media and physicochemical parameters used for cultivation of the recombinant yeast.
The milk-clotting activity and speci c activity of the transformed crude enzyme increased as the fermentation time increased.However, the total protein content of the crude enzyme did not show signi cant differences at the time of incubation increased.However, there is a slight increase in protein concentration for the crude enzyme extracted from the control on the 6 th day (Table 3.1, Fig. 3.5).Comparatively, a slightly higher protein concentration from recombinant yeast was noticed at 96 h of fermentation time.Similarly, the cultivation of recombinant P. pastoris under optimized conditions produced a maximum protein concentration at 72 h.Likewise, the cultivation of recombinant P. pastoris under optimized conditions produced maximum protein concentration at 72 h (Yegin and Fernandez-Lahore, 2013).

SDS-PAGE analysis
The SDS-PAGE analysis showed that the major protein expressed by recombinant yeast P. pastoris X-33 AP has a molecular mass between 32 and 46 kDa (Fig. 3.6B).However, some other proteins with molecular mass above 80 kDa were also observed on the SDS-PAGE.This may infer to be either from the proteins contained in the media, or some other proteins expressed apart from the protein of interest.
However, the recombinant aspartic proteinase expressed in P. pastoris with a molecular mass of 52.4 kDa (Sun et al. 2018) and46-58 kDa (Yegin andFernandez-Lahore, 2013) by SDS-PAGE was different from the present study.

Conclusions
Based on the results it can be concluded that the aspartic protease gene from Aspergillus oryzae DRDFS 13 cloned and in P. pastoris X-33 AP was a functionally active protein with signi cant milkclotting activity.Therefore, the milk-clotting protease extracted from the recombinant yeast may be a suitable candidate for application in cheese and other food industries.
modi ed version that involves the author's contribution to the study; and have agreed both to be personally accountable for the author's owncontributions and ensure that questions related to the accuracy or integrityof any part of the work, even ones in which the author was not personallyinvolved, are appropriately investigated, resolved and the resolutiondocumented in the literature.Representation of pGAPZαA vector including the site of the restriction enzymes XhoI and NotI used for cloning.
Figure 4 Schematic representation of the pMK-AP vector used for protein expression.

Table 3
*ND-milk clotting activity not determined within 40 min, STD: standard deviation, Mean: is average of two measurements, Different letters (a, b, c, d) designate signi cantly different means as determined by Duncan multiple mean comparison test (P<0.05).