Transcriptomic studies on the product stress response revealed that YCF1 is a beneficial factor for progesterone production in Yarrowia lipolytica

Abstract

Progesterone is a widely used therapeutic hormone and a common precursor for the synthesis of pharmaceutical steroids in both mammals and plants. It has been successfully produced in heterologous microorganisms, but the market demand has not been met. The low progesterone yield, possibly due to product stress, may put pressure on the growth of strains and limit product synthesis efficiency. In this study, key pathways and genes that cause changes in amino acid and lipid metabolism and protein transport were identified through omics analysis. The expression of transporters leads to increased progesterone production and alleviated growth inhibition. Two related genes (gene IDs: 2912325 and 2908366) encoding the transporters glpF and SNQ2 improved production by 29.2 % and 51.7 %, respectively. Isoenzymes of native and exogenous transporters were screened and overexpressed. YCF1 from Saccharomyces cerevisiae exhibited the greatest benefit, increasing progesterone synthesis by 69.6 %. Our findings help reveal the impact of product stress on cellular metabolism and processes, providing research directions and literature support for the synthesis of other products.

Keywords: Omics studies; Product stress response; Progesterone; Transporters; Yarrowia lipolytica.

Graphical abstract

Fig. 1

The impact of the progesterone stress response on cellular metabolism. The genes (Δ7-reductase, CYP11A1, ADR, ADX and 3β-HSD) are heterologous genes overexpressed in the progesterone synthesis pathway, while the gene ERG5 (shown in red) needs to be knocked out for the pathway's expression.

Fig. 2

The response of Y. lipolytica to progesterone stress is reflected mainly in amino acid metabolism, lipid metabolism, and transport. A. Profile of differential gene expression under progesterone stimulation. B. Functional categories of KEGG (left)- and GO (right)-annotated genes that were differentially expressed in response to 15 min of progesterone treatment. C. The highly enriched functional categories of the KEGG-annotated genes found to be induced and repressed after 15 min of progesterone treatment.

Fig. 3

Identification and overexpression of candidate genes. A. A Venn diagram. The circles represent the genes whose expression was upregulated or downregulated during the response period, and the numbers represent the number of DEGs. The overlapping regions represent differentially expressed genes that were present during two or three response periods. B. Combination of a Sankey diagram and a bubble chart. Functional enrichment of genes induced after 15 min of progesterone treatment and cellular processes associated with specific candidate genes. C. Transcriptional profiles of sterol synthesis, amino acid metabolism, lipid metabolism and transport under progesterone treatment. D. Effects of the overexpression of candidate genes on biomass accumulation and progesterone production. E. Effects of the overexpression of candidate genes on precursor production.

Fig. 4

Effects of the overexpression of endogenous transporter genes on biomass accumulation and the production of progesterone and its precursors. A. Final OD₆₀₀ and progesterone production of strains overexpressing heterologous transporters. B. Intracellular and extracellular campesterol production in heterologous transporter-overexpressing strains. The percentage is the proportion of extracellular campesterol. (The error bars for all the data represent the standard deviations calculated from triplicate experiments. Significance levels of Student's t-test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.)

Fig. 5

Identification and overexpression of exogenous transporters. A. The general phylogenetic topology of heterologous transporters. B. Final OD₆₀₀ and progesterone production of strains overexpressing heterologous transporters. C. Intracellular (purple) and extracellular (cherry red) campesterol production in heterologous transporter-overexpressing strains. The percentage is the proportion of extracellular campesterol. D. The effect of the transporter identified in this study on different progesterone-producing strains. E. Plane diagram of the molecular docking of progesterone with transporters.