An Interplay between Transcription Factors and Recombinant Protein Synthesis in Yarrowia lipolytica at Transcriptional and Functional Levels-The Global View

Abstract

Transcriptional regulatory networks (TRNs) associated with recombinant protein (rProt) synthesis in Yarrowia lipolytica are still under-described. Yet, it is foreseen that skillful manipulation with TRNs would enable global fine-tuning of the host strain's metabolism towards a high-level-producing phenotype. Our previous studies investigated the transcriptomes of Y. lipolytica strains overproducing biochemically different rProts and the functional impact of transcription factors (TFs) overexpression (OE) on rProt synthesis capacity in this species. Hence, much knowledge has been accumulated and deposited in public repositories. In this study, we combined both biological datasets and enriched them with further experimental data to investigate an interplay between TFs and rProts synthesis in Y. lipolytica at transcriptional and functional levels. Technically, the RNAseq datasets were extracted and re-analyzed for the TFs' expression profiles. Of the 140 TFs in Y. lipolytica, 87 TF-encoding genes were significantly deregulated in at least one of the strains. The expression profiles were juxtaposed against the rProt amounts from 125 strains co-overexpressing TF and rProt. In addition, several strains bearing knock-outs (KOs) in the TF loci were analyzed to get more insight into their actual involvement in rProt synthesis. Different profiles of the TFs' transcriptional deregulation and the impact of their OE or KO on rProts synthesis were observed, and new engineering targets were pointed.

Keywords: heterologous protein; omics data; protein production; transcriptional regulation; yeast.

Figure 1

Clustered heatmap of transcriptomics (five sets ‘transcriptomics’) and functional (two sets ‘phenotypes’) data for the 140 TFs analyzed. Each row represents a single TF-encoding gene. Data are color-coded according to the legends on the right. Top legend—transcriptomics (‘0’ and white denotes no change); bottom legend—phenotype (‘1’ and white denotes no change). Numbers in the cells denote the fold change value over the control strain. The lack of numbers indicates a lack of statistical significance. Ten clusters were delimited. In the figure, subsequent clusters are separated by a bold horizontal line. The heatmap was split between clusters 1 and 2 for clarity of presentation.

Figure 2

A subgroup of TFs assigned to a ‘cellular response to nutrient levels’ category. Data color-coding and mode of presentation are according to Figure 1, and a legend is presented there.

Figure 3

A subgroup of TFs displaying an inverted deregulation pattern in the transcriptomic data of HSS and UPR strains. Data color-coding and mode of presentation are according to Figure 1 and a legend presented there.

Figure 4

A subgroup of TFs displaying a uniform deregulation pattern in the transcriptomic data (at least 3 out of 5 datasets). Data color-coding and mode of presentation are according to Figure 1, and a legend is presented there. The correlation matrix for these data is shown in Figure S2.

Figure 5

Change in the rProt synthesis capacity investigated in direct comparative functional studies of strains bearing OE (navy) and KO (green) of a given TF. Top chart–fold change (FC) in the total amounts of rProt synthesized by the engineered strains over the control strain, read as fluorescence from the reporter protein. Bottom chart–fold change (FC) in the total amounts of rProt synthesized by the engineered strains over the control strain, read as fluorescence from the reporter protein and normalized per accumulated biomass (read as absorbance at 600 nm). The horizontal line at the level of 1.0 indicates the level of rProt by the control strain. Bars indicate mean values from 2 to 6 subclones cultivated in technical duplicate ± SD. Asterisks (*) indicate a statistically significant difference between the modified strain and the control strain at p < 0.05.