Investigation of Kluyveromyces marxianus as a novel host for large‐scale production of porcine parvovirus virus‐like particles

Abstract

Background Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines. Results After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice. Conclusions This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

A Single Vaccination of IBDV Subviral Particles Generated by Kluyveromyces marxianus Efficiently Protects Chickens against Novel Variant and Classical IBDV Strains

“…As a unicellular eukaryotic microbe, yeast is considered as a particularly powerful platform for SVPs/VLPs production because it possesses PTM, grows cheaply and rapidly, and readily ferments in high cell density [40]. There are a growing number of vaccines, with more than 30 types that have been produced in three yeast species, including Saccharomyces cerevisiae, P. pastoris, K. marxianus, and Hansenula polymorpha [21,41]. More importantly, production of IBDV SVPs in yeast such as K. marxianus and P. pastoris reaches more than 1.0 g/L, which is significantly higher than in B/IC and E. coli, since the expression levels in these two hosts were lower than 100 mg/L with the maximum AGP titer of no more than 1:16 [34,35,42].…”

“…Kluyveromyces marxianus (K. marxianus) is one of the fastest-growing eukaryotic organisms that has been recognized as GRAS by the FDA, as well as QPS by the EFSA [19]. It is also a promising cell factory for the production of virus-like particles (VLPs) [20,21]. In this study, we used K. marxianus to express nvIBDV VP2 protein with or without an N-terminal His tag.…”

Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology

“…Spontaneously assembly of CAP protein into VLPs showed the prospects of cost-effective preparation of PCV2 vaccines. Recently, Yang et al (2021) used K. marxianus to evaluate the expression and downstream processing of PPV VLPs. The VP2 protein from Kresse strain was subjected to expression following optimization by the codon bias of K. marxianus .…”

Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus

“…K. marxianus is a natural thermotolerant yeast, which is a promising host for the production of cellulosic ethanol, chemicals and heterologous proteins [31][32][33][34]. In our previous study, the toxicity of Cre recombinase has been proved in K. marxianus [18].…”