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To enable the sustainable production of ovalbumin (OVA) without relying on animal sources, the generally recognized as safe (GRAS) host Saccharomyces cerevisiae was used for the precision fermentation-based production of recombinant OVA. For this purpose, a signal peptide derived from EPX1, the most abundant extracellular protein produced by Pichia pastoris, was identified as a novel signal peptide for the efficient secretion of OVA in S. cerevisiae. To improve OVA secretion and cell growth, three helper proteins (PDI1, KAR2, and HAC1) present in the endoplasmic reticulum were expressed individually or in combination. Notably, the +P1/K2 strain coexpressing PDI1 and KAR2 with OVA produced 2 mg/L of OVA in the medium fraction; this value was 2.6-fold higher than the corresponding value for the control strain without helper proteins. Finally, a glucose-limited fed-batch fermentation process using the +P1/K2 strain yielded 132 mg/L of total OVA with 8 mg/L of extracellular OVA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L -1 in shake flasks using the OVA expression cassette containing a tac promoter (P tac ) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (P P5 combined with P tac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L -1 , respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L -1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5' side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production., (© 2023 Wiley-VCH GmbH.)

Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)