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.

Keywords: INU1; Inulinase; Kluyveromyces marxianus; MgSO4; Single-chain antibody (scFv).

Fig. 1

Design of INU1 disruption and replacement. a A schematic of the INU1 locus and its deletion is shown. Reverse complement sequences of target (red) and PAM (shadowed) are shown on the reference sequence. The lower part of the panel shows an alignment of the sequence from the deletion strain with the sequence of the wild-type locus. b A schematic of the INU1 replacement by a fragment encoding scFv is shown. The mutated PAM sequence and its reverse complement sequence are highlighted in yellow. Mutated bases are indicated as small red letters. The scFv-encoding fragment also encodes the INU1 secretion signal peptide (blue box) and a 6-histidine tag (red box) in-frame with the expressed ORF

Fig. 2

INU1 deletion and replacement analyzed by agarose gel electrophoresis. Sequences from the transformed cells were PCR-amplified and analyzed by agarose gel electrophoresis. A pair of primers (P_Km01-010 + P_Km01-011) flanking the INU1 locus were used to amplify intervening sequences. The resulting amplicons from the wild-type strain (lane 1), inu1-deleted strain Δinu1 (Km02-063) (lane 2), or scFv integration strains No. 6 (Km02-064) (lane 3) and No. 7 (Km02-065) (lane 4) matched the expected fragment sizes of 3057, 492, 2184, and 2184 bp, respectively. DNA ladder markers (1 Kb and 100 bp) are provided as size standards (lanes M1 and M2, respectively)

Fig. 3

INU1 deletion analyzed by SDS-PAGE and secretion of scFv analyzed by immunoblotting. a Schematic of the general construction of scFv-expression plasmid. kanMX: G418 resistance gene, KmCEN-D: centromere sequence of K. marxianus, KmARS7: autonomous replication sequence of K. marxianus. b Combinations of promoters and signal peptides tested. c Stains were cultivated for 48 h in YPD with or without G418, and SDS-PAGE was used to analyze the spent culture medium from strains No. 1 (wild-type) (lane 1), No. 6 (Km02-064) (lane 2), No. 7 (Km02-065) (lane 3), and No. 8 (Km02-066) (lane 4). Sizes of the molecular weight marker (lane M) are indicated on the left. d Immunoblot of spent culture medium from strains cultivated for 72 h in YPD with or without G418. Lane numbers correspond to those in panel (b)

Fig. 4

Immunoreactivity of secreted scFv analyzed by ELISA. Strains were cultured in YPD or YPX in the absence or presence of MgSO₄ at 20 °C (a) or 30 °C (b). Spent culture medium was harvested at 72 h and subjected to ELISA using lysozyme as an antigen for scFv. Values are presented as mean ± SEM from three independent experiments