Improvement of recombinant L-Asparaginase production in Pichia pastoris.

Pichia pastoris is a successful expression system that is frequently preferred in the secretion of proteins for both basic research and industrial purposes. In this study, recombinant Rhizomucor miehei (RmASNase) L-asparaginase was produced in Pichia pastoris. The impact of gene copy number on increasing protein production was examined with six clones harboring various gene copy numbers (1-5 and 5 +). The results demonstrated that the clone with three copies of the expression cassette integrated had the highest production level. Also, biochemical characterization of the enzyme was performed. It was determined that the optimum pH and temperature values of the purified enzyme were pH 7.0 and 50 °C, respectively. Stability analyses of the enzyme showed that it maintains its activity of 80% in the pH range of 5-9 and 67% in the temperature range of 20-50 °C. Ca+2 and Mn+2 ions increased the enzyme activity to 121% and 138%, respectively. In future studies, it is also possible to improve the activity and stability values of the enzyme with advanced molecular techniques and to increase production efficiency by producing at fermenter scale and under optimum conditions.

Applicability of the heterologous yeast promoters for recombinant protein production in Pichia pastoris.

Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters (Kluyveromyces marxianus TPI, Hansenula polymorpha PMA, Candida tropicalis ICL, and Saccharomyces cerevisiae CUP) in Pichia pastoris. The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI, glucose for CUP, and ethanol for ICL. The DNA region showing the highest activity was determined as 1000 bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters. KEY POINTS: • Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host. • HpPMA has the highest promoter activity among the heterologous promoters tested. • Reporter gene expression with ScCUP is responsive to elevating Cu2+in P. pastoris.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801 bp, and -284 and -108 bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262 bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.

Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris.

Pectinase is one of the most widely used enzymes in different fields of food industry for different purposes, such as clarification of fruit juice, extraction of vegetable oil and saccharification of agricultural substrates. The aim of this study is to produce recombinant pectinase and evaluate the effect of codon optimization and promoter selection on production. In this study, the gene encoding pectinase in Aspergillus niger was optimized according to the codon usage of Pichia pastoris. Within the scope of the study, codon-optimized and native (non-codon-optimized) pectinase genes were transferred to P. pastoris X33 strain and expressed under the regulation of methanol-inducible AOX1 and ethanol-inducible ADH2 promoter. As a result of the study, the promoter and codon combination which exhibited the highest production level was determined as the ADH2 and codon-optimized pectinase yielding an enzyme activity of 42.33 U/mL. The best producer clone was cultured in 400 mL media in 2 L shake-flask and the enzyme was purified by His-tag method. The optimum working conditions of the purified enzyme was 50 °C and pH 5.0, and after incubation at 60 °C for 1 -h, enzyme activity was maintained at 60% level. The Michelis-Menten constant (Km) and maximal velocity (Vmax) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 µmol/mg/min, respectively.

Identification of major ADH genes in ethanol metabolism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.

Cloning and expression of pullulanase from Bacillus subtilis BK07 and PY22 in Pichia pastoris.

In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40 °C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest activity at pH 8. BK07pul and PY22pul activities were determined as 8.46 U/mL and 15 U/mL. The enzyme stability of BK07pul was higher (89%) than PY22pul (68%) where relative activity was determined as activity remaining after 1 h at corresponding optimum conditions for each. Amino acid homology evaluation revealed the two enzymes had 80% identity in primary structure. The presence of conserved sequences consisting of 7 amino acids (YNWGYDP) in both enzymes confirmed these to be type I pullulanases, capable of hydrolyzing α-1,6 glucosidic bonds of pullulan resulting in maltotriose units.