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. 2019 Nov 29;18(1):207.
doi: 10.1186/s12934-019-1256-0.

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

Xueyun Zheng  1   2 , Yimin Zhang  1   2 , Xinying Zhang  1   2 , Cheng Li  1   2 , Xiaoxiao Liu  1   2 , Ying Lin  3   4 , Shuli Liang  5   6
Affiliations

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

Xueyun Zheng et al. Microb Cell Fact. .

Abstract

Background: The 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.

Results: The 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.

Conclusion: This 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.

Keywords: Enhanced protein production; Fhl1p; Pichia pastoris; Transcription factor; Translation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Fhl1p protein sequence comparison. Alignment of amino sequences of P. pastoris of Fhl1p and S. cerevisiae Fhl1p by the DNAMAN software. The amino acids were shown by one-letter codes. Gaps were introduced to maximize the similarity. In P. pastoris, the FH domain between amino acids 372 and 461 and its homologous region were marked by a black frame. A red line below the alignment was used to mark the FH positions of S. cerevisiae. An asterisk character was used to indicate positions that have a single, fully conserved residue
Fig. 2
Fig. 2
Characterization of the Fhl1p function on promoting the expression of recombinant protein. a Phytase expression levels when Fhl1p was overexpressed in the 6 phy strain. b Pectinase expression levels when Fhl1p was overexpressed in the 4 pel strain. c mRFP expression levels when Fhl1p was overexpressed in mRFP
Fig. 3
Fig. 3
Analysis of differential gene expression of the 4 pel/AF in comparison to 4 pel strain. Red arrows (↑) indicate an increasing and green arrows (↓) indicate a decreasing of the corresponding transcriptional levels in the methanol induction phase
Fig. 4
Fig. 4
Polysome profiles and (M + P):(40S + 60S) ratios for strains grown in methanol conditions. a Representative polysome profiles and b a chart presenting (M + P):(40S + 60S) ratios of the different strains. Corresponding peaks (40S, 60S, 80S/monosomes and polysomes) are indicated in the polysome profile. (M + P):(40S + 60S) ratios were calculated from areas beneath the profile curve using ImageJ
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