Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica—molecular background revealed by quantitative proteomics

Abstract

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg−1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo–keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided. Key points • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica. • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation. • Global proteome under Osm, Temp and Osm Temp treatments was studied.

Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load

“…Abundance of necessary transcription factors, polymerase elements, nucleotides and others were secured by higher rate of the nutrients consumption ( Figure 1 , Table 1 ) that could be obtained at a higher oxygen transfer capacity (i.e., kLa 110 h −1 ). Similar results were obtained by [ 22 ], where the Authors observed lower expression levels of a native lipase in Y. lipolytica cultured under oxygen deficiency. In addition, the critical role of OA for the expression level of heterologous genes in Y. lipolytica is further highlighted by the lack of significant impact of the medium pH on the heterologous gene transcription level ( Figure 2 ).…”

“…The model strains were subjected to a series of batch cultivations with implemented stress factors, namely acidic pH and limited OA, applied alone or in combination. Although Y. lipolytica is known to grow well in a wide pH range without relevant impact on growth [ 17 , 28 , 29 , 33 , 34 ], oxygen deficiency is a strong limiting factor for this species [ 18 , 19 , 22 , 28 ]. Combination of the extrinsic stress factors and metabolic load in relation to growth, morphology and rs-Prots synthesis has not been studied previously in Y. lipolytica .…”

“…As obligate respiratory organism, a critical factor that is technically challenging in Y. lipolytica -based rs-Prots production process is oxygen availability (OA) [ 18 , 19 , 20 ]. Sufficient supply of oxygen was found to be the key factor affecting production capacity of multiple target products (enzymes and metabolites) by Y. lipolytica , precluding high efficiency of the corresponding process [ 21 , 22 , 23 , 24 , 25 ]. We recently reported that a volumetric oxygen transfer coefficient (kLa) lower than 50 h −1 (i.e., low OA) negatively affects cell growth and carbon source uptake rate as compared to values above 100 h −1 (high OA) [ 19 ].…”

“…The effect of various environmental conditions on the synthesis of native and recombinant proteins in Y. lipolytica has been addressed in several previous works. With the aim to study the impact of local gradients in pH and OA encountered in large-scale bioreactor, a scaled-down approach was used to investigate individual responses awakened by limited OA and fluctuations in pH, implemented independently [ 22 ]. It was found that insufficient OA had a pronounced effect on the gene expression and ultimately on the synthesis of the corresponding rs-Prot, while the simulated pH gradient was of less significant impact.…”

“Fight-flight-or-freeze” – how Yarrowia lipolytica responds to stress at molecular level?

“…Downregulation of membrane potassium channel (A00847p) hallmarks active adjustment of ions equilibrium. Another downregulated protein was sphingolipid long-chain base-responsive protein Lsp1 (C11341p), which together with Pil1 (identified as osmo-stress responsive in Y. lipolytica (Kubiak-Szymendera et al 2021b )) are known to act in close proximity of cell membrane, and to be involved in the regulation of cell growth, heat stress responses, endocytosis, wall synthesis and repair, as well as repolarization of the actin cytoskeleton in response to stresses. Further insight into the Y. lipolytica osmo-proteome was provided in a recent study by Kubiak-Szymendera et al ( 2021b ).…”