Biomedical applications of yeasts - a patent view, part two: era of humanized yeasts and expanded applications

ABSTRACT

Introduction

Yeast humanization, ranging from a simple point mutation to substitution of yeast gene(s) or even a complete pathway by human counterparts has enormously expanded yeast biomedical applications.

Areas covered

General and patent-oriented insights into the application of native and humanized yeasts for production of human glycoproteins (gps) and antibodies (Abs), toxicity/mutagenicity assays, treatments of gastrointestinal (GI) disorders and potential drug delivery as a probiotic (with emphasis on Saccharomyces bulardii) and studies on human diseases/cancers and screening effective drugs.

Expert opinion

Humanized yeasts cover the classical advantageous features of a ‘microbial eukaryote’ together with advanced human cellular processes. These unique characteristics would permit their use in the production of functional and stable therapeutic gps and Abs in lower prices compared to mammalian (CHO) production-based systems. Availability of yeasts humanized for cytochrome P450 s will expand their application in metabolism-related chemical toxicity assays. Engineered S. bulardii for expression of human proteins might expand its application by synergistically combining the probiotic activity with the treatment of metabolic diseases such as phenylketonuria via GI-delivery. Yeast models of human diseases will facilitate rapid functional/phenotypic characterization of the disease-producing mutant genes and screening of the therapeutic compounds using yeast-based high-throughput research techniques (Yeast one/two hybrid systems) and viability assays.

KEYWORDS:

• Yeast
• functional genomics
• high-throughput assays
• autophagy
• genome engineering
• glycosylation
• mannosyl-transferase
• human–yeast cross-species complementation
• yeast deletion collection
• saccharomyces boulardii

Article highlights

• Despite similarities of yeasts to higher eukaryotes, several critical differences limited their widespread utilizations that is relieved by ‘yeast humanization’ approaches.

• ‘Yeast humanization’ could be any change(s)/modification(s) in yeast ranging from ‘a simple point mutation to substitution of yeast gene(s) or even exchange of a complete pathway with human counterparts.’

• Due to critical N/O-glycosylation differences, most of the recombinant therapeutic proteins (RTPs) are produced in mammalian cells (CHO) rather than yeasts. Humanization of the yeast glycosylation pathways is changing the scenario in favor of yeasts for the production of high yield, functional RTPs with lowered cost.

• Humanization of N-glycosylation involves inactivation (gene knockout) of yeast OCH1 gene (encoding mannosyl-transferase) and/or replacement by their human-type counterparts and/or those enzymes responsible for the introduction of human-specific sialylated-biantennary sugar chains and terminal galactosylation.

• Humanization of O-glycosylation involves the inactivation of yeast encoding O-Mannosyl transferase gene(s) (PMT1-6) and introduction of GalNAc-T encoding genes to enhance mammalian mucin-type O-glycosylation of proteins.

• Natural yeasts are successfully used for the production of Ab fragments lacking a glycosylated Fc domain (e.g.: scFv), but for full-length Abs with glycosylated Fc domains, yeasts humanized for glycosylation are being used.

• Natural and humanized yeasts are used for mutagenesis assays. The presence of endogenous cytochrome P450s (CYPs) and possibility of heterologous expression of mammalian/human CYPs in yeast (humanization) made this organism an invaluable model system for metabolism-related chemical toxicity.

• Saccharomyces boulardii, possesses properties of a perfect probiotic agent with adjuvant/anti-inflammatory/anti-toxin/antimicrobial effects in GI tracts and natural insensitiveness to prokaryotic-specific antibiotics (as a eukaryote). It also represents one of the most promising nano-particulate drug delivery carriers and systems.

• Recent progresses on heterologous expression of RTPs in S. boulardii have enhanced the idea of synergistically combining the probiotic activity of this yeast with additional health benefits to treat metabolic diseases such as phenylketonuria (ingestion of S. boulardii encoding human phenylalanine ammonia Lyase).

• Unique features of yeast such as haploid maintenance (ease of the phenotype observation), ease of diploid cell formation by mating, sharing conserved and orthologous genes and similar cellular structure/functions to human cells have made yeast a perfect model for human diseases/cancers. Identification of autophagy in yeast (awarded the 2016 Nobel Prize) facilitated the screening of protein aggregation/misfolding toxicity associated with several degenerative diseases.

• Yeast expression of mutant disease-associated human proteins (or yeast mutant-ortologs) combined with ‘yeast-based high-throughput research techniques (Y1H and Y2H) and viability assays’ has resulted in screening for valuable therapeutic compounds against human diseases/cancers.

• For the production of RTPs, humanized P. pastoris is the favored yeast because of higher yields while humanized S. cerevisiae is the most addressed yeast for yeast-based models and assays.

This box summarizes key points contained in the article.

Author contributions

FR was involved in the conception, design, organization, literature hunting and final drafting, revising and editing of all sections and final approval of the version to be published. PE was involved in the design, literature hunting and primary drafting of sections 2 and 6 (‘Humanization of yeast-derived recombinant glycoproteins’ and ‘Native and humanized yeasts as model systems for human diseases’). MA-A was involved in the design, literature hunting, and primary drafting of section 3 and 5 (‘Production of recombinant antibodies by native and humanized Yeasts’ and ‘Yeast as a probiotic and ingredient of functional foods’). MS was involved in literature hunting and organization of the citations and assisted in the primary drafting of section 4 (Native and humanized Yeasts as model systems for toxicity and mutagenicity screening). NK was involved in literature hunting and editing of the parts related to the ‘human diseases’ and ‘therapies’ throughout the manuscript. All authors agree to be accountable for all aspects of the work.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work was supported in part by grant number 849 entitled, ‘Construction of whole yeast vaccine encoding HPV-E7 antigen’ from Pasteur Institute of Iran.