Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus

Abstract

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (β-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man 3 GlcNAc 2 and the human complex N-glycan Man 3 GlcNAc 4 , where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

“…Recent contributions to the toolbox of genetic parts for tunable gene expression in K. marxianus include the characterization of native and non-native promoters, terminators, selection markers, and low- and high-copy origins of replication ( Kumar et al, 2021 ; Lang et al, 2020 ; McTaggart et al, 2019 ; Rajkumar et al, 2019 ; Yang et al, 2015 ). In addition, CRISPR-Cas9 systems have been published by five groups and successfully applied for the synthesis of several compounds, including fatty acids ( Cernak et al, 2018 ), 2-phenylethanol ( Li et al, 2021 ; Rajkumar and Morrissey, 2020 ), ethyl acetate ( Löbs et al, 2017 , 2018 ), human N-glycan structures ( Lee et al, 2020 ), and a single-chain antibody ( Nambu-Nishida et al, 2018 ). A detailed summary of the current K. marxianus CRISPR systems can be found in Table S1 .…”

A novel ALG10/TGF-β positive regulatory loop contributes to the stemness of colorectal cancer

“…The successful cell surface TGFBR2 transportation requires both complex and oligomannosidic type [21]. ALG family is an important family of mannosyltransferases, for example, ALG3 holds α-1,3-mannosyltransferase activity and trigger the biosynthesis of lumen oligosaccharide, this is necessary for N-linked glycosylation [26]. And ALG3 distortion leads to the loss of mannose in oligosaccharide chain.…”

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation

“…A review of these efforts is provided by Laukens et al (2015). Another recent study used CRISPR/Cas9 to humanize the yeast Kluyveromyces marxianus (Lee et al, 2020). The success of these efforts suggest that some IgG biotherapeutics could eventually be produced using yeast expression systems.…”