Recombinant expression and characterization of Oryctolagus cuniculus chymosin in Komagataella phaffii (Pichia pastoris).

This study was conducted for investigating expression and enzymatic characteristics of recombinant Oryctolagus cuniculus chymosin (ROCC) expressed in Pichia pastoris. SDS-PAGE of partially purified supernatant displayed two distinct molecular bands approximately at the sizes of 40 kDa and 45 kDa corresponding to chymosin and partially glycosylated chymosin, respectively. Proteolysis assay demonstrated that rabbit chymosin was more specific compared to bovine and camel chymosins when it comes to hydrolyzing α, ß, and κ-casein. Rabbit chymosin kept its stability in a wide pH range (3.0-6.0) at 37 °C for 8 h. Active chymosin exhibited maximum enzymatic activity at 40 °C and pH 4.0 with the addition of 75 mM CaCl2. The ROCC clotting activity on donkey, cow, goat, lamb, camel milk was determined as 40, 10, 5.7, 3.07, and 2.66 IMCU/mL, respectively. These results revealed that ROCC might possess a potential for incorporation into cheese manufacture technology as a milk-clotting enzyme.

Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris.

Milk-clotting enzymes used in the dairy industry can be obtained from different sources such as plants, animals, and microorganisms. Recombinant chymosin is the best alternative for the dairy industry due to the differences in physicochemical properties of coagulating enzymes and scarcity of chymosin from animal sources. In this study, glycosylated and non-glycosylated forms of yak chymosin were extracellularly produced in a methylotrophic yeast, Komagataella phaffii (Pichia pastoris). Synthetic yak prochymosin genes were cloned into the pPICZαA vector, expressed in P. pastoris GS115 (PDI) strain. Active chymosin expression was achieved into supernatant with Saccharomyces cerevisiae α-mating factor under the control of methanol-inducible AOXI promoter. The glycosylation of yak chymosin did not have a significant effect on yield and activity at shake flask level. In a 5L fermentor, production of native yak-chymosin was achieved and the enzyme activity was found as 214 IMCU/ml. pH of 6-7 and temperature of 40 °C values were optimum for the enzyme. The laboratory scale white cheese production yield with recombinant yak chymosin was very similar to a commercial bovine chymosin. These results indicate that P. pastoris expression system is very suitable for recombinant yak chymosin production to meet the needs of the cheese industry.

Generation and characterization of caprine chymosin in corn seed.

Chymosin is widely used in the dairy industry, and much is produced through recombinant DNA in organisms such as bacteria and tobacco. In this study, we used a new transgenic method to express caprine chymosin in corn seeds with lower cost and better storage capability. The recombinant chymosin protein was successfully expressed at an average level of 0.37 mg/g dry weight, which is 0.27% of the total soluble protein in the corn seed. Prochymosin can be activated to produce a chymosin protein with the ability to induce clotting in milk, similar to the commercial protein. The activity of the purified recombinant chymosin was as high as 178.5 U/mg. These results indicate that we have successfully established a technology for generating corn seed-derived caprine chymosin for potential use in the dairy industry.

Expression and characterization of camel chymosin in Pichia pastoris.

Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45-50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese.

Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter.

A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01h(-1). By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37°C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.

Enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115.

Chymosin can specifically break down the Phe105-Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS-PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25-50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn(2+), Fe(3+), Mg(2+) and Na(+), but inhibited by K(+), Co(2+), Zn(2+), Ni(2+), and to a lesser extent by Cu(2+). These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

BACKGROUND: The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. RESULTS: Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. CONCLUSIONS: In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli.

BACKGROUND: Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes. The use of codon-optimized genes may overcome this problem. However, designing a gene for optimal expression requires choosing from a vast number of possible DNA sequences and different codon optimization methods have been used in the past decade. Here, a comparative study of the two most common methods is presented using calf prochymosin as a model. RESULTS: Seven sequences encoding calf prochymosin have been designed, two using the "one amino acid-one codon" method and five using a "codon randomization" strategy. When expressed in Escherichia coli, the variants optimized by the codon randomization approach produced significantly more proteins than the native sequence including one gene that produced an increase of 70% in the amount of prochymosin accumulated. On the other hand, no significant improvement in protein expression was observed for the variants designed with the one amino acid-one codon method. The use of codon-optimized sequences did not affect the quality of the recovered inclusion bodies. CONCLUSIONS: The results obtained in this study indicate that the codon randomization method is a superior strategy for codon optimization. A significant improvement in protein expression was obtained for the largely established process of chymosin production, showing the power of this strategy to reduce production costs of industrial enzymes in microbial hosts.

Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin.

BACKGROUND: Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL(-1) in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. RESULTS: Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid-containing and Ca(2+) -deficient medium, but Ca(2+) supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1-N-prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL(-1) in the K. lactis PMR1-deficient mutant, sixfold higher than that in wild-type K. lactis GG799. CONCLUSION: Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production.

[Gene synthesis of the bovine prochymosin gene and high-level expression in Kluyvermyces lactis].

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

Chymosin and other milk coagulants: sources and biotechnological interventions.

Calf rennet, which consists of over 90% chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

[Recombinant expression of bovine chymosin in Pichia pastoris].

To express bovine chymosin in yeast, we amplified the prochymosin gene from the plasmid pMD18T-Prochy by PCR, and then cloned the gene into the expression vector pPICZaA, resulting in pPICZaA-Prochy. Pichia pastoris GS115 was used as host cells. Integration of the prochymosin cDNA into the Pichia pastoris genome was confirmed by PCR and sequencing analysis. Chymosin was expressed in Pichia pastoris successfully, and a strong band at about 37 kD was shown by SDS-PAGE. Activity tests showed that the chymosin activity of the culture supernatant was 12.2 SU/mL. This is the first report of successful expression of chymosin in Pichia pastoris. The recombinant Pichia pastoris strain obtained in this study could be further used to produce recombinant chymosin for cheese making.

Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant.

Aspergillus oryzae has attracted much attention as a host for heterologous protein production because of its high secretion ability and safety. However, there have been only a few reports on construction of this organism to improve its properties as a production host. We previously reported that the double disruptant of the proteinase gene (tppA, pepE) improved human lysozyme (HLY) production. In this double disruptant, however, the HLY expression plasmid cannot be removed due to its random integration into the genome. In this study, we re-constructed the tppA pepE disruptant as a host for heterologous protein production. By the use of the tppA pepE disruptant, bovine chymosin (CHY) production was enhanced by 1.9-fold. Moreover, we generated HLY-producing strain from the tppA pepE disruptant by curable niaD marker, and then isolated HLY-hyperproducing mutants using the halo assay based on HLY activity. Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection. The cured strains (named AUT strains) showed production levels of HLY and CHY that were 2.6- and 3.2-fold higher than those of the wild-type strain, respectively. Thus, the AUT strains are expected to be good hosts for obtaining higher production levels of various heterologous proteins.

Increased production of chymosin by glycosylation.

Filamentous fungi are well known in the industry as producers of large amounts of extracellular proteins. However, production levels of heterologous proteins are often disappointing low. In this paper it is shown that increasing glycosylation is a powerful strategy for increasing production levels of chymosin in filamentous fungi. Two different concepts based on glycosylation were tested. First, we improved a poorly used N-glycosylation site within the prochymosin molecule. The resulting highly glycosylated chymosin molecule was expressed in Aspergillus niger. It was shown that production of the glycosylated protein was much more efficient, giving a yield increase of more than 100% compared to production of the native chymosin molecule. In an alternative strategy the N-glycosylation site was located outside of the native chymosin molecule, on a linker separating prochymosin from its carrier molecule. Also in this case significantly increased production levels were obtained. This strategy might offer a powerful tool for increasing production levels of other heterologous proteins as well.

Molecular cloning and expression in yeast of caprine prochymosin.

We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.

[Rennet production by Rhizomucor miehei NRRL 3169]. / Producción de renina por Rhizomucor miehei NRRL 3169.

The production of rennet was studied, using different strains of the fungus Rhizomucor miehei. The selection and preservation of strains, type of growth, media design and operation conditions were evaluated. The experiments were carried out in Erlenmeyer flasks in rotary shaker at 250 rpm and 2.5 eccentricity, and in mechanically stirred fermentors of the New Brunswick type, at 30 degrees C. In the studies concerning strain selection, the best strain was Rhizomucor miehei NRRL 3169. The major titles of enzyme were obtained in batch process at 168 h, with 884 SU/ml, whereas in mechanically stirred fermentors the best value was 1160 SU/ml. These values were far more superior to former ones published by various experts.

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences.

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.