Heterologous Expression of Extracellular Proteinase pAsPs of Aspergillus pseudotamarii in Komagataella phaffii

Abstract

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5–8.0, and optimal temperature—50–60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…methods; obtaining basic biochemical characteristics of pretarates such as dependence of enzyme activity on pH and temperature; and obtaining highly purified drugs using highpressure liquid chromatography methods [246,[330][331][332]. The cited studies also point to the high potential of K. phaffii for industrial applications in terms of its secretion capacity and some advantages from the standpoint of protein folding and glycosylation, which positively affect enzymes' properties and thermal stability.…”

“…Figure 4 shows the main stages of the process of obtaining recombinant enzyme-producing strains based on the K. phaffii genome and the experimental approaches used in cloning the target gene, including cloning the target gene into the genome; selection by assessing the obtained clones based on the activity of the target protein, the results of quantitative PCR analysis of its genes in the genome and the accumulation of its fraction in cultural fluid; semi-quantitative analysis of its content in the culture liquid by electrophoresis of the target protein; analysis accumulation of the biomass and protein during the cultivation of selected clones in a bioreactor; full genomic sequencing of the nucleotide sequence of the genome of the strain producing the target protein; identification of the amino acid sequence of the target protein in the process of expression and secretion of this gene by the yeast genome when cultivating the producer strain in a bioreactor; obtaining protein and enzyme preparations by tangential diffusion and liquid chromatography methods; obtaining basic biochemical characteristics of pretarates such as dependence of enzyme activity on pH and temperature; and obtaining highly purified drugs using high-pressure liquid chromatography methods [ 246 , 330 , 331 , 332 ].…”

“…So the data outlined above are evidence of the successful use of K. phaffii for the heterologous production of almost all the major enzymes important for increasing the nutritional value of feed, including the most popular ones: thermostable phytases [ 198 ], glucoamylases [ 229 , 230 , 231 , 232 , 233 , 234 ], and α-amylases [ 223 , 224 , 225 ], including the co-expression of glucoamylase and α-amylase genes [ 230 ] as well as for the heterologous synthesis of xylanase [ 246 , 247 , 253 ], mannanase [ 276 , 277 , 279 , 281 , 282 , 283 , 285 , 332 ], laccase [ 314 ], cellulases [ 316 ], and proteases [ 326 , 327 , 328 , 329 , 330 , 331 ].…”

“…An example is the K. phaffii strains secreting recombinant proteases from A. niger [ 326 , 327 , 328 ], Bispora sp. MEY-1 [ 329 ], Aspergillus pseudotamarii [ 330 ], or Tritirachium album [ 331 ].…”

“…( a ) Target gene cloning; ( b ) analysis of recombinant clones for the presence of protein and enzyme activity; ( c ) electrophoretic analysis of the mannanase preparation from the culture liquid of recombinant clone; ( d ) analysis accumulation of biomass and protein during cultivation of selected clones in a bioreactor; ( e ) full genomic sequencing of the nucleotide sequence of the genome of the strain producing the target protein; ( f ) analysis of the culture fluid of the producer line (general view of the MS1 mass spectrogram containing the peptide mixture obtained from the culture liquid); ( g ) the fragment peptide sequence founding in culture fluid samples of the recombinant strain K. phaffii T07 identifying the structure with the specified putative amino acid sequence with a convergence of 58.22%; ( h ) protein and enzyme preparations by tangential diffusion and liquid chromatography methods; ( i ) characteristics of purified and lyophilized proteinase K, dependence of enzyme activity on pH and temperature. Adapted from [ 246 , 330 , 331 , 332 ].…”