Hansenula: Versatility

Hansenula polymorpha for System Versatility

The extraordinary versatility of the Hansenula expression system lies in the ability to express multiple proteins within the same cell. A vector can be designed with two or more expression cassettes in a tandem arrangement; subsequent transformants will always have the genes present in equimolar ratios. This approach has been used successfully for the production of human hemoglobin. The different expression cassettes can also be placed in different plasmids and introduced in successive transformations. Further flexibility is thus derived from the ability to select the optimal gene ratio for the system under investigation. This opens up innumerable possibilities in both recombinant protein production and in biocatalysis. It is particularly well suited for engineering multiple-step bioconversions, since different enzymes can be expressed in the ratios leading to optimal product yield. The following examples highlight some of the recent successes using this system.

Hansenula as a Biocatalyst
Glycolate oxidase is a peroxisomal enzyme which has been used for the biocatalytic production of glyoxylic acid from glycolate. In order to avoid further oxidation of the glyoxylate product, the hydrogen peroxide generated during the reaction must be removed by a catalase. Hansenula polymorpha was engineered to perform this bioconversion by co-expression of spinach glycolate oxidase (GO) and S. cerevisiae catalase T (CTT1). A strain bearing approximately 30 copies of integrated plasmid with the GO gene behind the FMD promoter was transformed with a second plasmid bearing the CTT1 gene behind the same promoter. After selection by G418 resistance, strains with high CTT1 activity were scored for desired characteristics. CTT1 copy number ranged from 2-25 copies; all double-transformants showed excellent mitotic stability, with no detectable change in the integrated DNA after 800 generations of growth under non-selective conditions. The strain showing the highest of both enzymatic activities was selected for fermentation. The cells were harvested, permeabilized, and then used in a bioconversion reaction. When ethylenediamine was included in the reaction, yields of glyoxylate exceeded 97%. Cells could easily be recovered by centrifugation and reused in as many as 25 successive reactions.
Correct Processing of Recombinant Hirudin in Hansenula
	Hirudin is a potent and specific inhibitor of thrombin and has gathered considerable interest because of its potential as a therapeutic and/or diagnostic agent. To test the suitability of H. polymorpha as an expression host, the hirudin sequence was fused to the prepro segment of MFα1, GAM1 with an added KEX2 site, or CHH, each downstream of the MOX promoter. Selected strains were fermented, and the secreted hirudin was analyzed for the presence of variants by HPLC. All constructs directed the secretion of hirudin, and the protein synthesized with the GAM1 or CHH prepro sequence was authentically processed. When the MFα1 prepro sequence was used, approximately 13% of the hirudin had a 1 amino acid N-terminal extension. In all 3 cases the amount of full-length hirudin was 70-90% of the total secreted, depending on the fermentation condition. The species that normally contaminates full-length hirudin in S. cerevisiae could not be detected. BASF/Knoll AG is currently evaluating the H. polymorpha-derived hirudin in Phase III clinical trials.
*Use of Hansenula* for Hepatitis B Vaccine Production**
Serum-derived Hepatitis B particles consist of 90% S, 5% M, and 5% L surface antigens. Since the S-antigen has been shown to carry antigenic sites important for developing immune responses in humans, an effective vaccine composed of virus-like particles consisting of only the S-antigen was developed using the H. polymorpha expression system. High level intracellular expression of S-antigen particles was achieved using H. polymorpha strains that stably maintained several copies of the S-antigen expression plasmid integrated into the genome. The H. polymorpha-derived vaccine consisting of purified S-antigen particles has been produced by Korean Green Cross Corporation and has been approved for use in humans by the World Health Organization.

Designing Hansenula polymorpha as a Biocatalyst for the Oxidation of α-Hydroxy Acids

Two potential industrial applications of the enzyme glycolate oxidase (GO) are its use in converting glyoxylate to glycolic acid and pyruvate to lactate. Technical Application Note No. 101 summarizes a series of experiments examining an efficient biocatalytic process for glycolic acid and lactate production developed using a strain of Hansenula polymorpha which expresses high levels of spinach glycolate oxidase enzyme.

Note-101.pdf

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For more information about this Expression System visit www.artes-biotechnology.com

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