Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins

Abstract Background Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. Results To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. Conclusion These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast

Momentum transfer dependence of reflection electron energy loss spectra: theory and experiment

Measured reflection electron energy loss spectra excited by 2 keV electrons in Si are simulated by a Monte Carlo approach using the Chen–Kwei theory to calculate the position-dependent inverse inelastic mean free path. By comparing measured and calculated spectra, several prescriptions concerning the momentum transfer dependence of the material dielectric function are discussed. Our results lend support to the approximation (often taken with the only justification of making relations simpler) of neglecting the momentum transfer component normal to the surface in the surface region. Copyright © 2014 John Wiley & Sons, Ltd