Hansenula: Yield

Hansenula polymorpha for Maximum Yield

The growing demand for a microbial expression system that circumvents the complications associated with E. coli has led to the development of a proprietary expression system in the methylotrophic yeast Hansenula polymorpha. As the exclusive North American representative for ARTES Biotechnology, BTR now offers the power of this technology to its clients.

Methylotrophic Yeast

The four known genera of methylotrophic yeast (Hansenula, Pichia, Candida, and Torulopsis) share a common metabolic pathway that enables them to use methanol as a sole carbon source (see figure). In a transcriptionally regulated response to methanol induction, several of the enzymes are rapidly synthesized at high levels. Since the promoters controlling the expression of these genes are among the strongest and most strictly regulated yeast promoters, methylotrophic yeast have become very attractive as hosts for the industrial production of recombinant proteins. The cells themselves can be grown rapidly to high densities, and the level of product expression can be regulated by simple manipulation of the medium.

Key Distinctions Between H. polymorpha and P. pastoris

The two species of methylotrophic yeast that have been developed for commercial-scale recombinant protein production are Hansenula polymorpha and Pichia pastoris. Although these two species are close relatives, there are some important distinctions between the species and the expression systems developed in them that should be considered when selecting the system for a specific application. Enzymes of the P. pastoris methanol utilization pathway, including alcohol oxidase, are rapidly and strongly induced in response to methanol induction. In H. polymorpha, the enzymes can be induced either by methanol or by glycerol derepression. This appears to be a feature of the organism itself since the AOX1 promoter of P. pastoris (the analogue of the H. polymorpha MOX promoter) is induced by glycerol derepression in H. polymorpha but not in P. pastoris. This is particularly significant for applications involving large-scale production, since it circumvents the potential hazards associated with the use of methanol.

The plasmids used for expression in P. pastoris are designed for homologous recombination and integration at the AOX or HIS4 locus, whereas those used in H. polymorpha integrate by non-homologous recombination at random loci. The number of plasmid copies integrated in the P. pastoris system is limited, usually to <10, whereas up to 150 copies have been integrated using the H. polymorpha system. Since protein expression is correlated with gene dosage in these organisms, it is possible to achieve significantly higher expression in H. polymorpha despite the fact that the AOX promoter of P. pastoris is the strongest promoter. It is also possible to achieve more control over the level of production by regulating the copy number. Furthermore, when plasmids are integrated at the AOX locus, the gene encoding the alcohol oxidase is disrupted; once the cells are transferred to methanol their growth is significantly retarded. The growth rate of H. polymorpha remains rapid before and after induction. Finally, H. polymorpha is a more thermotolerant organism than is P. pastoris, easily withstanding temperatures up to 43°C.

Features of the Expression Vectors

Plasmids have been designed as shuttle vectors for propagation in E. coli and thus contain an ori sequence and a selection marker (typically the B-lactamase gene). Although the plasmids possess autonomously replicating sequences (ARS) for chromosomal-independent amplification, they are integrated into the H. polymorpha chromosome by non-homologous recombination.

The expression cassette itself consists of either the MOX or the FMD promoter, a multiple insertion site, and a MOX terminator. For proteins to be secreted, a variety of leader sequences is available; these include the S. cerevisiae MFα1, S. occidentalis GAM1, and Carcinus maenas hyperglycemic hormone sequences. Alternatively, proteins can be targeted to the peroxisomes by inserting the universal S/A/C-K/R/H-L tripeptide motif at the C-terminus. Once genes are integrated, they show extraordinary mitotic stability with no detectable loss or rearrangement of plasmid DNA after 800 generations of growth in the absence of selection. A number of nutritional and antibiotic markers are available for selection, as are the appropriate strain variants.

Productivity

As with any expression system, the product yield varies depending on the specific application. Expression levels of more than 10 g/L have been achieved with some products; typical yields range from 0.5 to several g/L. In addition to plasmid copy number, factors that can affect product yield include efficiency of secretion, protein stability, cell density, and some of the salient features of the organism itself. H. polymorpha is a very efficient secretor, although low amounts of endogenous proteins are secreted. The efficiency is somewhat dependent on the specific sequences of the leader and of the protein itself; thus, two identical constructs with different target genes may show different expression levels.

The ability to test a variety of leader sequences is an important feature of this system, because it allows for the optimization of the leader for each specific application. Hansenula cells can be grown quite rapidly to high cell densities (150 g/L, dry weight) with typical fermentation times of 100-150 hr. Generally, proteins secreted from H. polymorpha are unlikely to be exposed to significant proteolysis; there is virtually no evidence of C-terminal truncation of the type often encountered in S. cerevisiae.

Post-Translational Modifications

The glycosylation capability of all yeast, including H. polymorpha, is restricted to the high mannose type of structure. However, the hyperglycosylation observed in Saccharomyces (in which 40 or more mannose residues are added onto the core) is a very rare occurrence in H. polymorpha. The organism contains a signal protease with Kex2-like recognition sequence, and proteins are found to be efficiently and accurately cleaved upon secretion. For other enzymatic modifications that are not native to the host, it may be possible to engineer a strain expressing the desired enzyme as well as the product of interest.

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