Hansenula polymorpha (Biology and Applications) || Technical Enzyme Production and Whole-Cell Biocatalysis: Application of Hansenula polymorpha

255

15

Technical enzyme production and whole-cell biocatalysis:

application of Hansenula polymorpha

Adam Papendieck, Ulrike Dahlems, Cerd Gellissen

15.1Introduction

Enzymes have found important application in a variety of industrial settings. The

growing demand for industrial enzymes and whole-cell biocatalyst systems has

spurred a significant amount of biotechnological research and development. In an

effort to make these enzymes and biocatalysts more efficient, gene technology is

being applied in a number of different strategies:

(1) Research is being directed at lowering the production cost of industrial

enzymes and increasing their availability through heterologous production in

expression systems such as H. polymorpha (Aehle and Misset 1999; Gellissen

2000). As industrial enzymes are often required in mass quantities, it is

essential that they be produced for very low cost.

(2) New enzymes are continually being isolated, analyzed, and their respective

genes cloned. Genes for so-called extremozymes (enzymes capable of

tolerating harsh conditions) have been isolated from extremophilic micro-

organisms and expressed efficiently in recombinant expression systems, thus

making them available for cost-effective application in industry (Hough and

Danson 1999; Demirjian et al. 2001).

(3) The enzymes themselves are being modified through rapidly developing

techniques of protein engineering or directed evolution. As protein modeling/

prediction techniques improve, attempts to increase the stability and activity

of existing enzymes by directly modifying amino acid composition are being

met with increasing success (Griffiths and Tawfik 2000; Rubingh 1997).

These techniques of protein engineering have also been applied in the de novo

design of industrial proteins (Rubingh 1997).

(4) Advances in pathway engineering have made feasible the development of

efficient whole-cell biocatalysts (Chotani et al. 2000; Zaks 2001; Bull et al.

1999). Whereas isolated enzymes are particularly useful for hydrolysis or

isomerization reactions, whole-cell biocatalysts are capable of efficiently

Hansenula polymorpha: Biology and Applications. Edited by G. GellissenCopyright © 2002 WILEY-VCH Veriag GmbH, WeinheimISBN: 3-527-30341-3

256 15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha

regenerating co-factors required for organic synthesis. The ability of manyengineered bacteria, fungi and yeasts like H. polymorpha to maintain enzymesand cofactors at stoichiometrically precise ratios is making them increasinglyuseful components in industrial reactions (Schmid et al. 2001).

Currently, the worldwide market for industrial enzymes is at about $1.5 billion(Maister 2001). Figure 15.1 illustrates the major industrial sectors making up themarket for these enzymes. Technical enzymes used in the detergent and textileindustries, e.g., make up about 62% of the market, and enzymes used in the foodindustry represent about 32%. Demand is predicted to grow significantly as newenzymes are isolated and as the functionality and low-cost availability of existingenzymes are improved through heterologous production and techniques of proteinengineering. In this chapter we will review several important groups of industrialenzymes, their application in industry, the ongoing development of techniques fortheir structural modification and heterologous production, and finally examinesome case studies of industrial enzymes produced in H. polymorpha.

Beverages7%

Animal feed7%

Starch4%

Bakery5%

Dairy14%

Detergents49%

Textile13% Leather

1%

Fig. 15.1 The relative size of market segments for industrialenzymes (taken from Aehle and Misset 1999, based onsales data from 1995).

15.1 Introduction 257

Tab. 15.1 Application of industrial enzymes

Industry Enzyme Enzymatic reaction Application

1 Carbohydrate 1processing

Brewing

Baking

Dairy

Alcohol/spiritsproduction

Wine and fruitjuiceproduction

Candy/confections

Detergents

oc-, p-amylasesglucoamylasesglucoisomerases

oc-amylasesP-glucanasesproteases,glucoamylasespullulanases

a-amylasesproteasesglucoamylaseshexose oxidases

chymosin, rennetlipaseslactasescatalases

a-, P-amylasesglucoamylasesproteasespectinases

a-, p-amylasescellulasespectinasesp-glucanasesglucose oxidase

amylasesinvertases

alkaline proteaseslipasesamylasescellulases

1 starch breakdown(hydrolysis of glycosidicbonds), isomerization ofglucose to fructose

starch breakdownhydrolysis of glycosidic(bonds), glucan, dextroseand maltose breakdown

starch breakdown,protein and dextrinbreakdown

destabilization ofcasien, lipidmodifications, lactosebreakdown, peroxidebreakdown

starch breakdown,dextrin breakdown,degradation of proteinsand pectins

starch breakdown,cellulose and pectindegradation, glucanbreakdown, oxidant

breakdown of starchproducts, saccharosebreakdown

degradation of proteins,lipids and carbohydrates

1 production of glucose Iand fructose syrups,production of sugar

conversion of starchto sugar, malting,filtrationimprovement,stabilization andclarification, alcoholfree and light beer

dough handling,gluten modification,bread crustcharacteristics

cheese making andprocessing, milkthickening, aromaimprovements,increasingdigestibility,preservation

starch liquefactionand conversion tosugar, preservationand clarification

starch liquefactionand conversion tosugar, maceration andclarification, filtrationimprovement, aromastabilization

e.g., marzipanproduction

removal of protein-,fat- and starch-basedstains, restorationof fiber texture andcolor

continued


Tab. 15.1 continued

Industry

\ Animal feed

Enzyme

I phytasep-glucanases

Enzymatic reaction

1 degradation of phyticacid and glucan

Application

\ boost useable Iphosphate content offeeds, reduction ofp-glucans

Paper and pulp

Textiles

Tea and coffee

xylanases

amylasescellulases

hemicellulasespectinases

breakdown of xylan

breakdown of starchand cellulose

breakdown of pectinand cellulose

pulp bleaching aid

fiber preparation

extraction agent

15.2

Important groups of technical enzymes

A number of different groups of enzymes are currently being developed for avariety of industrial applications. Table 15.1 gives an overview of how enzymes arecurrently employed in industry.

15.2.1Amylases/glycosidases

Amylases are utilized for the breakdown of starches into simpler sugars. Typically,an a-amylase is used to degrade larger carbohydrates into maltodextrins. For furtherprocessing, a glucoamylase can be used to yield glucose, subsequently a p-amylasewill yield maltose, and a glucose isomerase will yield fructose. All of these enzymesare important in the food industry for the production of various types of sugarsyrups, as well as for starch breakdown in baking, brewing and alcohol production.In addition, oc-amylases are commonly employed as detergent additives for thetreatment of starch-based stains and in the textile and paper industries as a sizing/desizing agent. Genes for many of these amylases have been cloned and expressedwith high efficiency using alternative expression systems (see discussion of H.polymorpha-based production of glucoamylase below). The conventional industrialamylases are derived from various species of Bacillus and Aspergillus, although avariety of new sources are being exploited. Invertase is a p-fructosidase useful forthe modification of sweeteners, namely the conversion of inulin mixtures tofructose. An invertase gene from Saccharomyces cerevisiae has been efficientlyexpressed in both Pichia pastoris and H. polymorpha, the latter having a slightlyhigher stability (Acosta et al. 2000). Thermostable oc-amylases requiring a minimalcalcium concentration to maintain their conformation at higher temperatures havebeen derived from Bacillus licheniformis and Bacillus stearothermophilis (Vihinen andMantsala 1989). Hyperthermostable (Joyet et al. 1992; Chen et al. 1996) and pH-tolerant (Tierny et al. 1995) variants have also been produced through protein

15.2 Important groups of technical enzymes 259

engineering, and some progress has been made in completely eliminating theirdependence on calcium for stability, making them safer for use in food processing(Aehle and Misset 1999).

15.2.2

Cellulase and xyianase

In vivo, groups of cellulases act synergistically to break down cellulose, a linear,unbranched polymer made up of glucose residues bonded through i,4-(3-glucosidiclinkages, comprising the chief component of the cell walls of plants. Theseenzymes have become increasingly important in the textile industry, particularly asagents for "stonewashing" and color maintenance (biopolishing). The mostcommonly utilized commercial cellulases are endoglucanases which break celluloseinto (3-glucans. These endoglucanases are typically derived from bacteria (Bacillus)or fungi (Aspergillus) which tend to be more tolerant of high pH (Hoshino and Ito1997). Cost-effective expression of both bacterial and fungal cellulase genes hasbeen achieved using a variety of host organisms, including the native organisms aswell as heterologous expression in a number of microorganisms (Miiller et al. 1998;Villanueva et al. 2000). In order to improve enzyme functionality, a fusion proteinwith two cellulose-binding domains has been engineered (Linder et al. 1996). Thedouble binding domain was found to bind cellulose much more tightly that thesingle domain. For a review of cellulase protein engineering, see Schulein (2000).

Xylan is the major constituent of hemicellulose and one of the most abundantrenewable resources on earth. Xylanases are used in the pulp and paper industry asan agent which optimizes the effectiveness of the bleaching process, thusdecreasing the amount of harsh bleaching chemicals required. Research onxylanases has recently attracted attention due to their ability to hydrolyzing xyloseinto fermentable compounds for alternative fuel production. Alkaliphilic andthermophilic xylanases have been isolated from a variety of extremophilicorganisms, and a number of different xyianase genes have been cloned andexpressed heterologously in organisms such as E. coli, B. staerothermophilus, S.cerevisae, L plantarum and Pichia stipitis (Kulkarni et al. 1999). Protein engineeringhas been met with limited success, although there have been advances incrystallization and preliminary X-ray analyses (Teplitsky et al. 2000). For athorough review, see Kulkarni et al. (1999).

15.2.3

Proteases and peptidases

Proteases comprise perhaps the most diversely applied group of technical enzymes.They are useful in processes ranging from paper, leather and textile preparation todetergent and food applications (Aehle and Misset 1999). The laundry detergentindustry provides a particularly large market for industrial proteases as they areapplied in the removal of protein based-stains. As with amylases and cellulases, asignificant amount of research is currently going into the isolation and develop-


ment of proteases functional under alkaline conditions, extreme water tempera-tures and in the presence of organic solvents for use in detergents. Extremophilessuch as Antarctic Bacillus TA39 (Narinx et al. 1997) and Halobacterium halobium(Ryu et al. 1994) have been exploited for these purposes. The oxidative stability ofsome proteases have been increased by substituting, e.g., the oxidizable Metresidues with non-oxidizable Ser (Bott 1997), and their heterologous production hasbeen achieved in a wide variety of expression systems for price reduction.Chymosin, e.g., cleaves a peptide bond in casein and is the crucial enzymecoagulant in the manufacture of cheese. It is traditionally derived from animalrennets, but attempts to more efficiently produce chymosin in microorganismssuch as Kluyveromyces lactis (Swinkels et al. 1993) and Aspergillus (Bodie et al. 1994)have led to a number of new recombinant chymosins on the market.

15.2.4Upases

Lipases are water-soluble enzymes which change conformation when in contact at awater-lipid interface, exposing a hydrophobic binding region. They act at thisinterface to hydrolyze triglycerides into glycerol and fatty acids. Due to theiramphiphilic properties, they have become extremely useful components ofdetergents for degrading triglycerides in water-insoluble, lipid-based stains. Theyare also commonly employed by the food industry to modify the lipid compositionof various fats, oils and food products. Useful Upases have been derived fromorganisms such as Thermomyces lanuginosa, Rhizomucor miehei, Candida andPseudomonas, and heterologously expressed with increased efficiency in, e.g.,Bacillus, Aspergillus and H. polymorpha (Aehle and Misset 1999). The proteinstructure and amino acid composition of a number of lipases have been modified inan effort to increase stability (Misset 1997) and the tendency of the molecule toaggregate at the water-lipid interface (Okkels et al. 1996). Progress has also beenmade in removing the strong calcium binding sites and restabilizing the enzymesthrough interactions not involving metal ion binding (Strausberg et al. 1995).

15.2.5

Other important enzymes

A number of other families of enzymes have found application in today's industry.Examples include the phosphate-degrading enzyme phytase, which can be appliedas a feed additive (Mayer et al. 1999; see also discussion of phytase production in H.polymorpha below), a number of catalases important in biocatalytic reactions forperoxide degradation (see discussion ofS. cerevsiae catalase CTTi below), pectinaseswhich are utilized to break down pectins in wines and fruit juice (Siekstele et al.1999), and oxidoreductases such as the hexose oxidase gene from the red algaChondrus crispus, which is useful in dough preparation and has been expressedheterologously in H. polymorpha and P. pastoris (Hansen and Stougaard 1997,Poulsen and Bak H0strup 1998).

15.4 The application of H. polymorpha as an expression system 261

15.3

Pathway engineering and biocatalysis

Aside from the application of heterologous expression technologies and proteinengineering, important biotechnological advances in pathway engineering and whole-cell biocatalysis hold significant potential for use in industrial chemical reactions. Thewell-studied metabolic pathways of a number of microorganisms have been appliedfor the processing of valuable substrates in efficient, enantioselective reactions.

In a recent review of the use of pathway engineering in commercial chemicalproduction processes, Chotani et al. (2000) point out that efforts to optimize whole-cell biocatylsts through metabolic engineering are focused in four major areas:

(1) optimization of transcriptionally and allosterically regulated primary pathwaysfor the production of the target products

(2) the genetic disabling of competing metabolic pathways,(3) enhancing carbon commitment to the pathway of interest through genetic

modification of the central metabolism,(4) modification of secondary pathways to enhance energy metabolism and

availability of required enzymatic cofactors.

Improvements in fermentation technologies and the development of strains moretolerant of organic solvents have also played a significant role in enabling these newwhole-cell biocatalytic systems to reduce the cost and amount of time required forthe synthesis of target chemicals. In addition, whole-cell biocatalysts offer a highlyenantioselective method for the production of pharmaceutical and technicalchemicals. For example, niacin, 2-cyanopryrazine, and nicotine have been producedusing Achromobacter ocylosoxidans, Agrobacterium and Pseudomonas, respectively, aswhole-cell biocatalysts (Schmid 2001), and other whole-cell systems are underdevelopment (see Sect. 15.4 on H. polymorpha as a biocatalyst).

15.4

The application of H. polymorpha as an expression system for technical enzymes andas a whole-cell biocatalyst

In order to be useful in an industrial setting, an expression system must producethe desired protein or metabolite at high levels and with extreme efficiency. A widevariety of genes for technical enzymes have been expressed in H. polymorpha (Table15.2), and here we describe some case examples of its use as an expression systemfor the production of a number of these enzymes, as well as its application as anefficient whole-cell biocatalyst.

15.4.1Glucoamylase production

Glucoamylase from the amylolytic yeast Schwanniomyces occidentalis was the firstheterologous protein to be secreted from H. polymorpha (Gellissen et al. 1991). The


Tab. 15.2 Genes for useful technical enzymes expressed in H. polymorpha

Enzyme Origin Secretion Reference

\ Cellulase-I ICellulase-IICellulase (heat stable)Glucoamylase

Glucose oxidaseGlycolate oxidaseHexose oxidase

Invertase

Lipase IPhytaseXylanase I

I Aspergillus aculeatus \Humicola insolensthermophilic organismSell wanniomycesoccidentalisAspergillus nigerspinachChondrus crispus

Saccharomyces cerevisiae

Thermomyces lanuginousAspergillusHumicola insolens

yesyesno

yesyesno-

yes

yesyesyes

I Miiller et al. 1998Miiller et al. 1998unpublished data

Gellissen et al. 1991Hodgkins et al. 1993Gellissen et al. 1996unpublished dataHansen and Stougaard1997Rodriguez et al. 1996Acosta et al. 2000Miiller et al. 1998Mayer et al. 1999Miiller et al. 1998

1

S. occidentalis enzyme exists as a 138 kDa cell-associated form and is secreted as a146 kDa glycoprotein. It has several characteristics which make it attractive to thefood industry, namely its ability to hydrolyze starch to glucose and to be inactivatedunder pasteurization conditions. The GAMi gene encoding the enzyme wasexpressed using its genuine secretion leader sequence. The entire coding sequence,including the secretion leader, was inserted into a H. polymorpha expression vectorunder the control of the FMD promoter. Following transformation by the protoplastmethod described by Dohmen et al. (1991), colonies were screened for secretion ofactive glucoamylase, isolated and passaged on alternating cycles of rich andselective media, resulting in mitotically stable strains. The approximate copynumber of the expression cassettes integrated into the genomic DNA was analyzedby agarose gel electrophoresis. When transferred to nitrocellulose and hybridized toa labeled FMD promoter probe, two signals in similar electrophoretic positionswere observed, one for the original genomic single copy FMD gene and a secondoriginating from the heterologous FMD promoter/leader sequence fusion. Thesignal intensity of the integrated DNA in comparison to the intrinsic single-copycontrol was estimated by a series of dilutions, and transformants harboring 1-8copies of integrated plasmid DNA were identified.

Glucoamylase activity assays revealed that strain 1,35, which harbored four copiesof the integrated expression cassette, produced the highest enzyme titer, andfermentation studies were thus continued with this strain. Trial fermentations werecarried out using 3% glucose for cell growth, and subsequent FMD promoterderepression was induced by substituting the glucose with i% methanol and 0.4%glycerol. Enzyme secretion was found to be linearly correlated with cell density, andwhen grown to a dry weight of 100-130 g L^1 the yield of biologically active enzymewas 1.4 g L"1. Fermenter media was fractionated in a Sepharose CL 6B column, andfractions displaying enzyme activity were further purified by ion-exchange

ISA The application of H. polymorpha as an expression system 263

chromatography. Southern blot analysis indicated that the enzyme was found to besecreted from H. polymorpha as a 150 kDa glycoprotein, and following deglycosyla-tion with EndoH or PNGaseF, a protein of 135 kDa was obtained (Figure 15.2). Thisis approximately the same size as the cell-associated form in S. occidentalis. Noproteolytic activity was detected, and the glucoamylase could be produced andstored without significant degradation.

15.4.2

Production of a heat-stable cellulase

As described above, cellulases are important in the paper and textile industries forcellulose breakdown. H. polymorpha was used for the production of a 35 kDa heat-stablecellulase derived from a thermophilic organism. A fragment harboring the codingsequence of the enzyme was inserted into the standard pFMPT 121 expression vectoryielding pFPMT-cell, under the control of the FMD promoter. Transformation of H.polymorpha was then carried out according to Dohmen et al. (1991), and transformantswere passaged and subsequently screened for cellulose productivity by a Congo redplate assay (Figure 15.3). Copy number characterization was carried out in a mannersimilar to that described for glucoamylase above. Transformants contained between 10and 70 integrated cassettes. Cellulase was found to be deposited intracellularly, and anSDS-PAGE analysis revealed that the heterologously produced enzyme had amolecular weight identical to the enzyme standard. A particularly productive straincontaining 10 copies of the cellulase cassette was selected for fermentation studies at a10 L scale. Standard |>O2-controlled fermentation on 3% glycerol was conducted, andlater methanol was added for FMD promoter induction. An enzyme yield in the rangeof several g per L cellulase was determined after 85 h fermentation time.

In order to verify the thermostability of the recombinant cellulase, it wasincubated for 30 min at 70 °C. Subsequent activity assays revealed that the cellulasemaintained 80% of its original activity, and SDS-PAGE analysis indicated adramatic decrease in host-derived proteins, indicating a possible new approach toinitial product purification.

Fig. 15.2 Comparison of the wild-typeS. occidentalis glucoamylase to therecombinant enzyme secreted from H.polymorpha. (i and 2) WT glucoamylase from S. 15Q kDa

occidentalis, (3 and 4) S. occidentalisglucoamylase produced in H. polymorpha LR 9(pFMDHEGAM). (i and 4) without PNGasetreatment. (2 and 3) with PGNase treatment.The proteins were separated by 7.5% SDS-PAGEusing Pharmacia's Phastsystem andglycosylated proteins visualized by fuchsinestaining (Gellissen et al. 1991).


Fig. 15.3 Congo red plate assay for cellulaseactivity. Potential transformant strains areplated on YNB containing 1% glycerol andincubated overnight at 37 °C. Plates are thentop layered with boiling agar (7% agarose in50mM K2HPO4, i2.5mM citric acid pH 6.3,0.5% carboxymethyl cellulose) which rupturesthe cells and releases any intracellularcellulase. Cooled plates are incubatedovernight at 55 °C, stained with a 1% solution ofCongo red, and destained 3omin later with ai M NaCI solution. Zones of cellulase activityshow up yellow, and then darken from thecenter, on an otherwise red background.

15.4.3

High-efficiency production of phytase

H. polymorpha has been used in a particularly efficient process for the production ofphytase (Mayer et al. 1999)- Phytases, as previously mentioned, are enzymes whichrelease phosphate groups from phytic acid. Addition of the enzyme to animal feedreleases the phosphate from phytic acid, which is normally inaccessible tomonogastric animals like pigs. The animal is thus able to utilize phytic acid as anefficient source of phosphate, decreasing the amount of inorganic phosphatesecreted into the environment (Wodzinski and Ullah 1996).

Phytase-expressing H. polymorpha strains were constructed by inserting one ofthree phytase coding sequences (derived from three species of Aspergillus) intoexpression vector pFPMT 121 containing the FMD promoter and URAj gene fortransformant selection. Transformation of the standard RBn strain was doneessentially as described by Zurek et al. (1996). Subsequent supertransformationswith expression cassettes harboring a phytase gene and an antibiotic resistancegene for transformant selection yielded strains with up to 120 integrated copies ofthe phytase expression cassette. The heterologous phytase was found to be secretedinto the media and accounted for more than 97% of total secreted protein.Transformants were screened for productivity, and several strains were chosen forfermentation at a 10 L scale. A specialized fermentation process was then developedto achieve high levels of enzyme production using inexpensive media. Significantly,it was found that the use of glycerol in the initial batch phase was not required andcould be substituted with low-cost glucose without drastically effecting productyield. Derepression of the FMD promoter was brought about by glucose starvation(fermentation with minimal levels of continuously fed glucose), thus eliminatingthe use of not only glycerol, but also methanol (which is indispensable in the pro-duction fermentation processes of other methylotrophic yeasts). At a 2,000 L scale,fermentation with glucose as the sole carbon source for growth and promotercontrol led to high product yields and an 80% reduction in raw material costscompared to glycerol-based fermentation. Furthermore, the H. polymorpha strainswere found to maintain high levels of productivity through repeated fed-batch

15.4 The application of H. polymorpha as an expression system 265

cultivations (Figure 15.4). Large-scale fermentation supernatants were purifiedthrough a series of flocculation centrifugation, dead-end filtration and a final ultra-filtration, yielding a high-quality, highly concentrated product at a recovery rate ofup to 92% (Figure 15.5). Strains were found to produce recombinant phytase atlevels ranging up to 13.5 g L"1. This high level of product yield in combination withthe successful development of a methanol-free fermentation process based on in-expensive glucose illustrates how H. polymorpha can be applied as an extremely effi-cient and economically competitive production organism for a technical enzyme.

15.4.4

Biocatalytic conversion of glycolate to glyoxylic acid

As described above, H. polymorpha has been used successfully for the production ofheterologous proteins. Furthermore, the system has also been developed for the co-expression of multiple genes, as with the production of the S and L hepatitis Bantigens in a fixed, optimized ratio (Janowicz et al. 1991). The ability to easily re-transform H. polymorpha with expression cassettes harboring additional genesmakes the yeast an attractive candidate for use as a whole-cell biocatalyst. In thiscase, we describe the use of H. polymorpha as a biocatalyst for the conversion ofglycolate to glyoxylic acid through the co-expression of two enzymes. Thebiocatalytic system described here may also be used for the conversion of lactateto pyruvate.

Ceil DryWeight BO

(g/L)

Phytase

(9/L)

120

- 2

- 1

240 360 480 600 720 840 960

Time (h)

Fig. 15.4 Repeated fed-batch fermentation ofa phytase-producing H. polymorpha strain.+ = level of secreted phytase. O=cell dryweight. Glycerol was used as sole carbonsource. At the end of each cycle, i L of cellsuspension was left in the bioreactor and 41

fresh sterile medium was added. Glycerolfeeding was re-started immediately for eachcycle (yoogL^1 glycerol solution added at5g l_~ 1 for 12 h, then log L^1 until the end ofthe cycle) (Mayer et al. 1998).

266 15 Technical enzyme production and whole-cell biocatalysis: application ofHansenula polymorpha

1 2 3 4 5

200 kDa

Fig. 15.5 Characterization of heterologouslyproduced phytase: SDS-PAGE analysis offermentation supernatants (lanes 3 and 5) andthe soluble fraction of disrupted cells (lane 4)after the standard cultivation of a phytase-producing strain of H. polymorpha. Lane 2 ispurified phytase from H. polymorpha, lane 5 isan overload for visualization of other secretedproteins, and lane i is the molecular weightmarker (Mayer et al. 1999).

21

Glycolate oxidase (GO) (Volokita and Sommerville 1987) is an FMN-containingenzyme found in the peroxisomes of both plants and animals catalyzing theoxidation of glycolate and its derivatives to glyoxylate and related 2-0x0 acids (Figure15.6). The peroxide formed during this conversion can seriously inhibit theefficiency of the biocatalytic system, so the gene for an S. cerevisiae-derived catalaseTi (CTTi) was co-expressed with GO. Unlike H. polymorpha's native catalase, thisheterologous S.c. CTTi was not found to be inhibited by the addition ofethylenediamine (which is an important supplement to the reaction mixture, seebelow) and, therefore, functions as the main catalase (Gellissen et al. 1996).

Following procedures similar to those described above, H. polymorpha strain RBuwas transformed with pRBGAO harboring a cDNA sequence encoding spinach GOand S.c. URAj as a selection marker. A stable, productive strain containingapproximately 30 copies of the integrated GO expression cassette was subsequentlysupertransformed with pRBCATT harboring the S.c. CTTi and a Tn5-derivedkanamycin resistance gene as an additional selection marker, yielding transformantswith 2-25 integrated CTTi expression cassettes (Gellissen et al. 1996).

Supertransformants were screened for enzyme activity. The two recombinantenzymes are sorted to different subcellular compartments; catalase T is cytosolicwhereas glycolate oxidase is deposited in the peroxisome. In intitial fermentationtrial at a 10 L scale, a productivity of 280 U GO per g wet cells and 140,000 U CTTiper g wet cells was achieved by harvesting at a cell density of 80 g dry weight L"1 at72 h. Together these heterologous enzymes represented up to 25% of totalintracellular soluble protein. Prior to use as a catalyst, the cells were permeablizedand washed with buffer, rendering them metabolically inactive and opening theintracellular environment to the reaction substrates and reagents. Aqueous reactionmixtures typically contained glycolic acid (o.863M), ethylenediamine (0.788 M) toprevent over oxidation to oxalate, isoburyric acid (0.075) as an HPLC internalstandard and permeabilized H. polymorpha as biocatalyst. Carried out in a stirredreactor under oxygen pressure at a pH between 8.9 and 9.1 and a temperature

15.5 Conclusion 267

glycolateoxidase (GO)

R-CHOH-COOH—/^r > R-CO-COOH

H9O2 » H2O + 1/2 O2catalase T(CTT1)

Fig. 15.6 Biocatalytic conversion of a-hydroxy CTTi can be applied as biocatalysts for theacids to the corresponding 2-0x0 acids, and production of glyoxylate, pyruvate from glycolicthe breakdown of resulting peroxide. acid, and lactate, respectively.H. polymorpha strains expressing CO and

between 5 °C and 15 °C, these reactor batches resulted in over 99% conversion toglyoxylate. The amount of glyoxylic acid produced per gram of catalyst was found tobe within acceptable limits for potential application as a commercial scalebiocatalytic process (Table 15.3). Furthermore, the cells can easily be collected bycentrifugation after each reaction batch and recycled up to 25 times with littledecrease in biocatalytic activity or product yield.

15.5

Conclusion

The utility of H. polymorpha in the production of recombinant antigens and othertherapeutic proteins has been well established (see Chapters 12-14; Gellissen 2000;Janowicz et al. 1991). The H. polymorpha-based process examples described in thischapter include the efficient production of a food additive (glucoamylase), a

Tab. 15.3 Glyoxylate yields and enzyme activities for consecutive oxidation reactions. Consecutivebatch oxidation reactions of 0.75 M glycolic acid using recycled H. polymorpha strain 7.13.63.8expressing heterologous CO and C7T? genes as a whole-cell biocatalyst (see text for reactionconditions). Percentage recoveries of microbial glycolate oxidase and total catalase activites arebased on initial activities determined at the start of the first reaction. Recoveries of greater than100% are due to an increase in the permeability of the cells over time

Reactionnumber

1,234

Glyoxylater/o yield]

\ 98.898.899.8

100

CO/% recovery]

\ 139114106107

CT total/% recovery]

1 s,104110139

CTTi/CTtotal l%]

U736063

1


thermostable industrial enzyme (cellulase), and a feed additive (phytase), as well asa description of the yeast as a whole-cell biocatalyst for the conversion of glycolate toglyoxylic acid. H. polymorpha is thus not only well suited for production ofpharmaceutical proteins, but also combines high expression levels with cheapfermentation procedures, achieving the high level of efficiency particularly criticalin the production of industrial enzymes. As the range of practical applications forindustrial enzymes grows, and as techniques of microbial biocatalysis are perfected,H. polymorpha holds the potential to be an extremely effective biotechnological toolfor use in industrial sectors.

References 269

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Hansenula polymorpha (Biology and Applications) || Technical Enzyme Production and Whole-Cell Biocatalysis: Application of Hansenula polymorpha