Genetic and physiological basis for antibody production by Kluyveromyces marxianus

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 (P MDH1 or P ACO1 ) 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

MOESM1 of Genetic and physiological basis for antibody production by Kluyveromyces marxianus

Additional file 1: Table S1. Primers used in this study. Figure S1. Amino acid sequence of scFv. Figure S2. Sequence of codon optimized scFv fragment. Figure S3. Sequence of E02-014 plasmid. Figure S4. Sequence of KmPMDH1. Figure S5. Sequence of KmPACO1. Figure S6. Secreted scFv activity per cell amount