Genome-scale metabolic model analysis of Pichia pastoris for enhancing the production of S-adenosyl-L-methionine.

Komagataella phaffii, formerly Pichia pastoris (P. pastoris), is a promising methylotrophic yeast used in industry to produce recombinant protein and valuable metabolites. In this study, a genome-scale metabolic model (GEMs) was reconstructed and used to assess P. pastoris' metabolic capabilities for the production of S-adenosyl-L-methionine (AdoMet or SAM or SAMe) from individual carbon sources along with the addition of L-methionine. In a model-driven P. pastoris strain, the well-established genome-scale metabolic model iAUKM can be implemented to predict high valuable metabolite production. The model, iAUKM, was created by merging the previously published iMT1026 model and the draught model generated using Raven toolbox from the KEGG database which covered 2309 enzymatic reactions associated with 1033 metabolic genes and 1750 metabolites. The highly curated model was successful in capturing P. pastoris growth on various carbon sources, as well as AdoMet production under various growth conditions. Many overexpression gene targets for increasing AdoMet accumulation in the cell have been predicted for various carbon sources. Inorganic phosphatase (IPP) was one of the predicted overexpression targets as revealed from simulations using iAUKM. When IPP gene was integrated into P. pastoris, we found that AdoMet accumulation increased by 16% and 14% using glucose and glycerol as carbon sources, respectively. Our in silico results shed light on the factors limiting AdoMet production, as well as key pathways for rationalized engineering to increase AdoMet yield.

Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris.

Secretion efficiency of the 85-amino acid Sacchromyces cerevisiae alpha signal peptide and the 25-amino acid Candida antarctica lipase B signal (nsB) peptide were compared. Three reporter proteins used for the study are C. antarctica lipase A (CalA), lipase B (CalB) and hGMCSF. The copy number of recombinant α-CalB and nsB-CalB clones was determined by qPCR and clones with equivalent gene copies were used for comparative analysis. About threefold increased CalB production corresponding to an activity of 480 U ml(-1) was obtained with its native signal peptide, whereas with the alpha signal peptide the maximum activity was 160 U ml(-1). Also, CalB was secreted as a mature protein with native N-terminus when fused to its own signal peptide, while unprocessed CalB with N-terminal extension was detected with the alpha signal peptide. Real time PCR analysis of CalB strains indicated that the difference in protein expression was not at the transcriptional level. The nsB signal sequence was also effective in secreting CalA enzyme and its secretion efficiency was on par with the alpha signal sequence. Further, hGMCSF fused inframe with the nsB signal peptide was also efficiently secreted into the medium. These results indicate that the nsB signal peptide can be a better alternative to alpha signal peptide for heterologous protein expression in Pichia pastoris.