Abstract
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.
Highlights
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
In this study, we demonstrated that K. marxianus NBRC1777 can be engineered to express and secrete a single-chain antibody
As the INU1 product inulinase metabolizes inuline to fructose (Rouwenhorst et al 1988), INU1 is downregulated in the presence of glucose, the preferred sugar (Jain et al 2012)
Summary
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. The inu1 deletion strain was generated by using a Cas9 plasmid (E02-026) containing gRNA-1 and gRNA-2 target sequences (Table 2 and Fig. 1a).
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