Microbial protein production

Apart from the application in biosensors A. adeninivorans is an excellent host for the production of recombinant proteins like human receptors. However, since some recombinant proteins require specific post-transcriptional and/or post-translational modifications the Xplor®2 transformation/expression platform was further developed to work with a wide range of yeast strains. Now additional yeasts like Hansenula polymorpha, Pichia pastoris, Kluyveromyces lactis, Yarrowia lipolytica, Debaryomyces hansenii, Debaryomyces polymorphus and Schwanniomyces occidentalis can be transformed and exploited as producers of recombinant proteins. The first functional recombinant receptors (e.g. human estrogen receptor α and human progesterone receptor) are synthesised at present for application in environmental analytics and, furthermore, in medical examinations. In the near future a surface plasmon resonance (SPR) platform for cancer analysis (e.g. breast cancer-interaction of Herceptin with the receptor HER2 [new]) and testing of novel drug-receptor-interactions will be developed in collaboration with the Fraunhofer Institute and industrial partners. 

 

Transgenic yeasts, especially A. adeninivorans are also used for the following biotechnological applications:

Tannase enzyme as supplement of animal feeds and/or as additive for the increased yield of biogas plants

Tannase producing transgenic A. adeninivorans strains are already available and tested in fed- batch fermentation trials. The achieved amount of tannase of 51900 U L-1 goes far beyond yields reached with other wild type yeasts or fungi so far. The transgenic Arxula strain was handed over to the ASA Spezialenzyme GmbH in Wolfenbüttel for commercial production of tannase.

 

Production of food with decreased purine content using an enzymatic procedure

Food with decreased purine content is of particular interest for patients suffering from gout. In order to establish an enzymatic procedure all genes of the purine degradation pathway (purinnucleotide phosphorylase, adenine deaminase, xanthine oxidase, guanine deaminase, urate oxidase) have been isolated and over expressed in A. adeninivorans in a transgenic approach to make the recombinant enzymes available in high concentrations. The first yeast strains for the production of adenine deaminase, urate oxidase and guanine deaminase were handed over to the ASA Spezialenzyme GmbH in Wolfenbüttel for commercial production. The strains for the production of purinnucleotide phosphorylase and xanthine oxidase are currently in process.

 

Application of A. adeninivorans as biocatalyst for the production of n-butanol

During the 1920's to 1940's n-butanol was already used as alternative fuel. With the shortage of crude oil and increasing prices of petrol alternative fuels are in great demand. Due to its energy value n-butanol represents a better option than currently available green fuels with ethanol additive. The problem is the availability of n-butanol which is produced so far by bacteria under strictly anaerobic conditions. Due to the low butanol tolerance of the bacteria the maximum yield is approximately 3% and, furthermore, the by-product acetate can hardly be separated from n-butanol. Because of its general robustness and putative tolerance to butanol and the availability of the complete genome sequence information as well as the ease genetically manipulation the yeast A. adeninivorans was selected as a potential biocatalyst for the production of n-butanol. After transformation of all bacterial genes necessary for the butanol synthesis a first transgenic yeast strain produced small amounts of n-butanol under not yet optimised cultivation conditions. After pathway and cultivation improvement it is expected to produce n-butanol in commercial scale. In order to ensure a fast conversion of the laboratory scale to a commercial application industrial partners (ACS Agrochemische Systeme GmbH/Homburg, Jäckering Mühlen- und Nährmittelwerke GmbH/Hamm) are involved in the work. Since the fermentation process is carried out under so-called semi anaerobic conditions in order to provide a sufficient amount of co-enzyme for n-butanol synthesis the cultivation conditions require particular optimisation which is done by biotechnologists of the Hochschule Anhalt in Köthen.

 

 

Production of polyhydroxyalkanoates PHB and P-(HB-HV) by Arxula adeninivorans

 

The production of biobased, biodegradable plastics has become increasingly interesting for research, not only because of the undesired accumulation of non-biodegradable plastics in the environment, but also because of the potential for new industrial applications. Above all, the polyhydroxyalkanoates poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate-co-hydroxyvalerate) (P-(HB-HV)) are commonly investigated, the latter being tougher and less brittle. The production of these polymers in bacteria is well established, but production in yeast has benefits, e.g. the ability to use a broad spectrum of industrial by-products as carbon sources.

We have increased the synthesis of P-(HB-HV) in A. adeninivorans by stabilization of polymer accumulation via genetic modification and optimization of culture conditions. An A. adeninivorans strain with overexpressed PHA pathway genes for β-ketothiolase, acetoacetyl-CoA reductase, PHA synthase and the phasin gene was able to accumulate a very high level of polymer. We found that an optimised strain cultivated in a shaking incubator is able to produce up to 52.1% of the dry cell weight of P-(HB-HV) with 12.3% mol of PHV fraction. Gel permeation chromatography analysis of the product revealed that MN (number average molar mass) and MW (weight average molar mass) values were 8,630 and 17,300 respectively which is considered to be very low. Additionally, we have applied the Differential scanning calorimetry (DSC) method to determine melting point (Tm) and glass transition (Tg) temperatures.

Our industrial partner Jäckering Mühlen- und Nährmittelwerke GmbH/Hamm has already built a pilot plant for the planned production of P-(HB-HV) copolymers. The factory building's infrastructure makes it possible to use substrates that are by-products of industrial starch production. The technical equipment of the plant has also already been procured and installed.

Conversion of Hydroxymethylfurfural to 2,5-Furandicarboxylic acid

 

2,5-Furandicarboxylic acid (FDCA) is a biobased alternative to petrochemically produced terephthalic acid, which is used for the production of polyethylene terephthalate (PET) and polyesters for the packaging and textile industry. Thus, there is a large market for this platform chemical. To date, no economic process for the production of FDCA is known, neither on a chemical nor on a biotechnological basis.

Our focus is on the use of safety-relevant biological systems based on the non-pathogenic yeasts Arxula adeninivorans and Hansenula polymorpha. We want to equip these with tailor-made enzymes that catalyse the reaction to FDCA from the raw substrate Hydroxymethylfurfural. Two approaches are being pursued: Firstly, the microbial synthesis of an intracellular bacterial HMF oxidase (HMFO) and secondly the synthesis of a genetically engineered aryl alcohol oxidase (AAOm).

After amplification, expression modules (TEF1 promoter - HMFO-/AAOm genes - PHO5 terminator) of the selected or mutated HMFO and AAOm genes are constructed and integrated into the genomes of A. adeninivorans and H. polymorpha using the Xplor®2 transformation/expression platform via "Yeast rDNA Integrative Expression Cassettes" (YRCs). The generated mitotically stable transgenic yeast strains are then analysed for accumulation of active recombinant AAOm or HMFO. The strains with the highest enzymatic activities (AAOm - extracellular, HMFO - intracellular) are then optimised for cultivation conditions to achieve maximum AAOm or HMFO accumulations.

Both enzymes will be investigated in both free and immobilised form. At the same time, the intracellular HMFO will also be used in whole cell catalysis, which would eliminate the need for complex enzyme purification.