Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. cloning of two novel promoters from Yarrowia lipolytica

Abstract

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

The methylotrophic yeast Hansenula polymorpha: a versatile cell factory

“…heterogenous glycosylation by addition of more than 40 mannose residues to the glycosylation core). In general, the overall length of the mannose chains is lower in H. polymorpha compared to S. cerevisiae and only few cases of hyperglycosylation have been reported in H. polymorpha [50,51]. Hence, H. polymorpha may be a much better candidate to produce human therapeutic proteins compared to S. cerevisiae.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…The most important problem is low ethanol yield and productivity from xylose in wild-type strains, which could be improved by classical selection and metabolic engineering (Kurylenko et al, 2014). It was previously confirmed that this yeast was able to produce high volumetric levels of recombinant endoglucanases derived from other fungi such as A. aculeatus and Humicola insolens (Müller et al 1998). A thermostable endoglucanase was also successfully produced in this yeast (Papendieck et al 2002).…”

Cloning of an epoxide hydrolase‐encoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica

“…The mono-integrative vector, pINA1313 (Nicaud et al, 2002), was modified by replacing the quasiconstitutive hp4d promoter (Madzak et al, 2000) with the TEF promoter (described by Müller et al, 1998). This replacement still allows high level expression of the cloned EH-encoding gene (driven by the TEF promoter), but with less growth phase dependence, as observed with the hp4d promoter.…”

“…Yarrowia lipolytica has been shown to be one of the most promising yeasts for heterologous protein production (Müller et al, 1998) and has been used for the production of recombinant proteins from a wide variety of organisms (Madzak et al, 2004). The Y. lipolytica system allows for the use of a wide variety of selective markers, promoters and secretion signals and stable site-directed or random integration of either single-or multi-copy expression cassettes into the genome.…”