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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.

Escherichia coli is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic E. coli strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of lpp (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.

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 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. [ABSTRACT FROM AUTHOR]