Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

Abstract

BackgroundThe methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins.ResultsThe function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities.ConclusionThis study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

“…Our first step was to find a proper P. pastoris strain. Although the strain P. pastoris GS115 was reported as a robust host to express recombinant proteins (Gurramkonda et al, 2009;Nie et al, 2014;Zheng et al, 2019;Wang et al, 2020), cell extracts prepared from this strain showed relatively little protein synthesis activity with very low and unreliable yields of sfGFP (data not shown). Then, we switched to the strain P. pastoris SMD1163 (pep4 prb1), which is derived from GS115 with knocking out of two protease genes (pep4 encodes protease A and prb1 encodes protease B) (Gleeson et al, 1998).…”

Heterologous and efficient expression of a new alkaline pectin lyase in Pichia pastoris

“…In a previous study in our laboratory, Zheng et al also proposed a novel yeast transcription factor, tentatively named Fhl1p, implicated in the regulation of rRNA processing genes and genes related to ribosomal small/large subunit biogenesis genes. This factor was found to facilitate the translation e ciency of exogenous proteins (Zheng et al 2019).…”

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

“…Consequently, the modulation of expression of genes encoding transcriptional regulators offers a variety of alternatives enabling an increment in the rProt production yield. However, the high number of genes affected by the modulated expression of these factors should be considered [76,79,103]. Given that, in general, the comparisons are performed under optimised process conditions for the wild-type strain, the results obtained by adapting the bioprocess to other transcriptional variants of K. phaffii are unknown [23].…”