Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris.

IMPORTANCE: Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Screening and evaluation of the strong endogenous promoters in Pichia pastoris.

BACKGROUND: Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. RESULTS: Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and ß-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the ß-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous ß-carotene biosynthetic pathway in P. pastoris, and the highest yield of ß-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. CONCLUSIONS: The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.