Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production

Abstract

Chymosin (EC3.4.23.4), primarily sourced from calf abomasum, serves as a conventional coagulant in milk curdling during cheese production. To improve the enzymatic properties and enhance the expression of calf chymosin (BtChy) in Kluyveromyces lactis to meet the demands of the cheese industry, the in silico engineering via hotspot scanning and molecular dynamics analysis was adopted. This approach improved the activity of BtChy on milk curdling and increased its sensitivity at 65°C. Multiple strategies were utilized to develop an environmentally friendly method for chymosin production. These included screening for constitutive promoters and signal peptides, as well as in vitro construction of a concatemer of the BtChy gene. The optimal combination, comprising the P_TDH3 promoter, invertase signal peptide, and a four-copy BtChy gene integrated into the yeast genome, was identified. After high-density cultivation in a 5-L bioreactor, the recombinant yeast achieved an activity of 42,000 SU/mL, a 52.5-fold increase over the original wild-type chymosin gene.

Keywords: calf chymosin; gene dosage; in silico engineering; promoter; signal peptide.

Figure 1

Displays the interactions between the amino acids in the active site of chymosin BtChy, its mutants, and the substrates. The residues located within a 4 Å radius adjacent to the cleavage site are highlighted. (A) The profile of the interaction between the chymosin and κ-casein. (B–H) The interaction details between the κ-casein and the active sites of wild type chymosin (WT) and the mutants M2, M3, M4, M5, M6, and M7, respectively. The mutant of M1 happened in pro-sequence of chymosin, and not be illustrated.

Figure 2

Point mutation of BtChy to improve its activity. (A) The diagram indicates the mutant sites and protein profiles of the recombinants checked by SDS-PAGE. (B) Chymosin activity of BtChy and its mutants.

Figure 3

Improving the secretory expression of BtChy by increase the gene-dosage in the genome of Kluyveromyces lactis by in vitro constructing the concatemers of expression cassettes. (A) Schematic diagram of in vitro construction the concatemers of expression concatemers of BtChy. (B) Checking the size of the concatemers of expression cassettes. (C) SDS-PAGE checking the supernatant of the culture of BtChy recombinants. (D) The chymosin enzyme activity of the supernatant of BtChy recombinants carrying different copy number of expression cassettes.

Figure 4

The energy variation RMSD and RMSF analysis of the wild type and mutant chymosin. The molecular dynamics simulation conditions are that the protein is placed in a 1 nm small cube box using an OPLS4 force field, and the water solvent model in the box uses TIP3P water molecule solvent; The system first uses the conjugate gradient method to minimize energy in 1000 steps; Under NVT conditions, the system is subjected to controlled heating from 0 to 303.15 K, with an integration step size of 1 fs and a duration of 100 ps; Then perform a 50 ps equilibrium simulation on the system for a duration of 100 ns. (A,C) RMSD and RMSF analysis of wild typy (WT) and mutant chymosin at room temperature. (B,D) RMSD and RMSF analysis of wild type (WT) and mutant chymosin at 65°C.

Figure 5

The optimal temperature of wild type and mutated BtChy. (A,B) The activity curves of enzymes incubated in different temperature. (C,D) The remaining activity when enzyme incubated under 65°C.

Figure 6

Promoter and culture carbon source screening for the expression of BtChy. Five constitutive promoters, P_GAP, P_TDH3, P_GCW14, P_PGK1, and P_SOR1 were used to control the transcription of BtChy. The yeast recombinants were cultivated in YPD medium with glucose, glycerol, sorbitol and methanol as the carbon sources. (A) The profiles of BtChy expressed and controlled by constitutive promoters, and cultivated in different carbon sources. (B) The chymosin activity of the recombinants culture.

Figure 7

Signal peptide screening to improve the secretory expression of BtChy. The full-length signal peptide of α-mating factor and truncated (α-MF, T-MF), α-amylase signal sequence (Amy), Inulinase presequence (Inu), serum albumin signal (HSA), invertase signal sequence (Inv), lysozyme signal peptide (Lys), glucoamylase signal peptide (Glu), and killer protein signal peptide (Kil) were used. The protein profile in the culture were detected by SDS-PAGE (A), and the activity of the recombinant protein were checked correspondently (B).

Figure 8

Production of chymosin BtChy in a 50-L bioreactor with high-density cultivation. (A) The protein profiles of supernatant checked by SDS-PAGE. (B) The curdling of milk after inoculates chymosin. (C) The protein standard curve for protein quantitation. (D) Protein content in the supernatant of culture. (E) The cell fresh weight of the culture and the enzyme activity measured.

Figure 9

Molecular dynamics analysis of cleavage distance between chymosin and κ-casein according to the double acid reaction and the hydrogen band analysis. (A) Mutant M2 and wild type chymosin (WT). (B) Mutant M6 and wild type chymosin (WT). (C) The hydrogen band network between chymosin M6 and κ-casein.