Engineering heterologous enzyme secretion in Yarrowia lipolytica

Abstract

Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and $$\alpha$$ α -amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

Comparative analysis of codon usage patterns of Plasmodium helical interspersed subtelomeric (PHIST) proteins

“…CUB has a significant impact on various cellular processes, including mRNA stability, transcription, translation efficiency and accuracy, protein structure, folding, expression, and function. Understanding CUB has practical applications in heterologous gene expression, species identification, primer design, predicting gene expression levels and functions, as well as designing synthetic genes for biotechnological purposes ( Gorlov et al, 2018 ; Chassalevris et al, 2020 ; Wang and Blenner, 2022 ; Hernandez-Alias et al, 2023 ; Vaz et al, 2023 ). However, most studies on CUB have focused on bacteria, fungi, viruses, and mycoplasma ( Dilucca et al, 2020 ; Hou, 2020 ; Wu et al, 2021 ; Li et al, 2022 ).…”

Analysis of Codon Usage Bias of Thioredoxin in Apicomplexan Protozoa

“…CUB is known to have a signi cant impact on a wide range of cellular processes such as mRNA stability, transcription, translation e ciency and accuracy, as well as protein structure, folding, expression, and function. Additionally, there are various practical applications in understanding CUB that are of signi cant value, including heterologous gene expression [19], identifying species origins [6,20], designing degenerate primers [21], predicting gene expression levels [22][23], predicting gene functions [24][25], and designing synthetic genes for biotechnological applications [26]. However, most of the numerous studies on CUB have focused on bacterium, fungi, viruses, and mycoplasma [27][28][29][30][31].…”