Genetic and physiological basis for antibody production by <italic>Kluyveromyces marxianus</italic>.

<italic>Kluyveromyces marxianus</italic> is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus <italic>K. marxianus</italic> has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered <italic>K. marxianus</italic> 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 (P_{<italic>MDH1</italic>} or P_{<italic>ACO1</italic>}) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type <italic>K. marxianus</italic> secretes endogenous inulinase predominantly, the corresponding <italic>INU1</italic> 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 <italic>INU1</italic> 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 <italic>K. marxianus</italic> has the potential to serve as a host strain for antibody production. [ABSTRACT FROM AUTHOR]