Date Published: April 12, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Yumiko Nambu-Nishida, Keiji Nishida, Tomohisa Hasunuma, Akihiko Kondo.
Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (PMDH1 or PACO1) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)—associated protein (CRISPR–Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv; the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.
Production of biopharmaceuticals requires the difficult choice of a host cell capable of generating the desired product in an active and safe form, devoid of unwanted modification or contamination. Additionally, some biopharmaceuticals such as antibodies have proven difficult to express at high levels. Chinese hamster ovary (CHO) cells and yeasts are the major hosts that have been engineered to produce biopharmaceutical products, including antibodies (Maccani et al. 2014). As mammalian cells, CHO cells produce mammalian-derived proteins in an active form, bearing appropriate modifications such as glycosylation. However, the development of stable cell lines takes very long times (6–12 months), and the cost of cell culture is very high (Lai et al. 2013). Bacterial expression host such as Escherichia coli provides much cheaper option, while proteins that require eukaryotic post-translational modifications are not suitable (Swartz 2001; Jevševar et al. 2005). Yeast cells such as Pichia pastoris may provide much faster and cheaper ways of production (Çelik and Çalık 2012); while this yeast can be engineered to serve as a suitable hosts, highly complex proteins such as antibodies can be difficult to express efficiently in this system (Nielsen 2013). In the previous study, we have shown that Kluyveromyces marxianus grow faster than Saccharomyces cerevisiae at wider range of temperature (Nambu-Nishida et al. 2017). K. marxianus also does not show obligate ethanol production aerobically and thus is expected to be engineered to produce various substrates (Wagner and Alper 2016).
In this study, we demonstrated that K. marxianus NBRC1777 can be engineered to express and secrete a single-chain antibody. We showed that secretion of scFv could be changed substantially by use of various genetic constructs and by modification of the growth conditions.