Production of bovine beta-lactoglobulin and hen egg ovalbumin by Trichoderma reesei using precision fermentation technology and testing of their techno-functional properties.

The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming. Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40-70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

Structure and function of aldopentose catabolism enzymes involved in oxidative non-phosphorylative pathways.

Platform chemicals and polymer precursors can be produced via enzymatic pathways starting from lignocellulosic waste materials. The hemicellulose fraction of lignocellulose contains aldopentose sugars, such as D-xylose and L-arabinose, which can be enzymatically converted into various biobased products by microbial non-phosphorylated oxidative pathways. The Weimberg and Dahms pathways convert pentose sugars into α-ketoglutarate, or pyruvate and glycolaldehyde, respectively, which then serve as precursors for further conversion into a wide range of industrial products. In this review, we summarize the known three-dimensional structures of the enzymes involved in oxidative non-phosphorylative pathways of pentose catabolism. Key structural features and reaction mechanisms of a diverse set of enzymes responsible for the catalytic steps in the reactions are analysed and discussed.

Three-dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6-bladed ß-propeller hydrolase family.

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d-xylonolactone. We have determined crystal structures of Cc XylC in complex with d-xylonolactone isomer analogues d-xylopyranose and (r)-(+)-4-hydroxy-2-pyrrolidinone at high resolution. Cc XylC has a 6-bladed ß-propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+ . The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+ . The structure of Cc XylC was compared with eight other ester hydrolases of the ß-propeller hydrolase family. The previously published crystal structures of other ß-propeller hydrolases contain either Ca2+ , Mg2+ , or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.

Xylonolactonase from Caulobacter crescentus Is a Mononuclear Nonheme Iron Hydrolase.

Caulobacter crescentus xylonolactonase (Cc XylC, EC 3.1.1.68) catalyzes an intramolecular ester bond hydrolysis over a nonenzymatic acid/base catalysis. Cc XylC is a member of the SMP30 protein family, whose members have previously been reported to be active in the presence of bivalent metal ions, such as Ca2+, Zn2+, and Mg2+. By native mass spectrometry, we studied the binding of several bivalent metal ions to Cc XylC and observed that it binds only one of them, namely, the Fe2+ cation, specifically and with a high affinity (Kd = 0.5 µM), pointing out that Cc XylC is a mononuclear iron protein. We propose that bivalent metal cations also promote the reaction nonenzymatically by stabilizing a short-lived bicyclic intermediate on the lactone isomerization reaction. An analysis of the reaction kinetics showed that Cc XylC complexed with Fe2+ can speed up the hydrolysis of d-xylono-1,4-lactone by 100-fold and that of d-glucono-1,5-lactone by 10-fold as compared to the nonenzymatic reaction. To our knowledge, this is the first discovery of a nonheme mononuclear iron-binding enzyme that catalyzes an ester bond hydrolysis reaction.

Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact.

Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C-C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning-guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. KEY POINTS: • DERA aldolases are versatile biocatalysts able to make new C-C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts.

Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods.

In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. KEY POINTS: • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.

Bioconjugation with Aminoalkylhydrazine for Efficient Mass Spectrometry-Based Detection of Small Carbonyl Compounds.

Bioconjugation through oxime or hydrazone formation is a versatile strategy for covalent labeling of biomolecules in vitro and in vivo. In this work, a mass spectrometry-based method was developed for the bioconjugation of small carbonyl compounds (CCs) with an aminoalkylhydrazine to form stable hydrazone conjugates that are readily detectable with electrospray ionization mass spectrometry (ESI-MS). Out of all hydrazine reagents tested, 2-(dimethylamino)ethylhydrazine (DMAEH) was selected for further analysis due to the fastest reaction rates observed. A thorough study of the reaction kinetics between structurally varied short-chain CCs and DMAEH was performed with the second-order reaction rate constants spanning in the range of 0.23-208 M-1 s-1. In general, small aldehydes reacted faster than the corresponding ketones. Moreover, a successful reaction monitoring of a deoxyribose-5-phosphate aldolase-catalyzed reversible retro-aldol cleavage of deoxyribose was demonstrated. Thus, the developed method shows potential also for ESI-MS-based enzyme kinetics studies.

In vitro reconstitution and characterisation of the oxidative D-xylose pathway for production of organic acids and alcohols.

The oxidative D-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from D-xylose or D-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of D-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family D-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family D-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.

Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica.

BACKGROUND: Crude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate. RESULTS: To prevent methanol conversion to CO2 via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from Yarrowia lipolytica. The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δfld1 strain. CONCLUSIONS: In silico, the expression of heterologous formaldehyde assimilation pathways enable Y. lipolytica to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable Y. lipolytica to efficiently use methanol as co-substrate with glycerol.

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family.

The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.7 Å resolution and compare it with other available enzyme structures from the IlvD/EDD protein family. The quaternary structure of CcXyDHT is a tetramer, and each monomer is composed of two domains in which the N-terminal domain forms a binding site for a [2Fe-2S] cluster and a Mg2+ ion. The active site is located at the monomer-monomer interface and contains residues from both the N-terminal recognition helix and the C-terminus of the dimeric counterpart. The active site also contains a conserved Ser490, which probably acts as a base in catalysis. Importantly, the cysteines that participate in the binding and formation of the [2Fe-2S] cluster are not all conserved within the Ilv/ED dehydratase family, which suggests that some members of the IlvD/EDD family may bind different types of [Fe-S] clusters.

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family.

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.

The Crystal Structure of a Bacterial l-Arabinonate Dehydratase Contains a [2Fe-2S] Cluster.

We present a novel crystal structure of the IlvD/EDD family enzyme, l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.1.25), which catalyzes the conversion of l-arabinonate to 2-dehydro-3-deoxy-l-arabinonate. The enzyme is a tetramer consisting of a dimer of dimers, where each monomer is composed of two domains. The active site contains a catalytically important [2Fe-2S] cluster and Mg2+ ion and is buried between two domains, and also at the dimer interface. The active site Lys129 was found to be carbamylated. Ser480 and Thr482 were shown to be essential residues for catalysis, and the S480A mutant structure showed an unexpected open conformation in which the active site was more accessible for the substrate. This structure showed the partial binding of l-arabinonate, which allowed us to suggest that the alkoxide ion form of the Ser480 side chain functions as a base and the [2Fe-2S] cluster functions as a Lewis acid in the elimination reaction.

Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Šresolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, ß = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Šresolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, ß = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%.

Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases.

We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg(2+) for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.

Characterization of a unique Caulobacter crescentus aldose-aldose oxidoreductase having dual activities.

We describe here the characterization of a novel enzyme called aldose-aldose oxidoreductase (Cc AAOR; EC 1.1.99) from Caulobacter crescentus. The Cc AAOR exists in solution as a dimer, belongs to the Gfo/Idh/MocA family and shows homology with the glucose-fructose oxidoreductase from Zymomonas mobilis. However, unlike other known members of this protein family, Cc AAOR is specific for aldose sugars and can be in the same catalytic cycle both oxidise and reduce a panel of monosaccharides at the C1 position, producing in each case the corresponding aldonolactone and alditol, respectively. Cc AAOR contains a tightly-bound nicotinamide cofactor, which is regenerated in this oxidation-reduction cycle. The highest oxidation activity was detected on D-glucose but significant activity was also observed on D-xylose, L-arabinose and D-galactose, revealing that both hexose and pentose sugars are accepted as substrates by Cc AAOR. The configuration at the C2 and C3 positions of the saccharides was shown to be especially important for the substrate binding. Interestingly, besides monosaccharides, Cc AAOR can also oxidise a range of 1,4-linked oligosaccharides having aldose unit at the reducing end, such as lactose, malto- and cello-oligosaccharides as well as xylotetraose. (1)H NMR used to monitor the oxidation and reduction reaction simultaneously, demonstrated that although D-glucose has the highest affinity and is also oxidised most efficiently by Cc AAOR, the reduction of D-glucose is clearly not as efficient. For the overall reaction catalysed by Cc AAOR, the L-arabinose, D-xylose and D-galactose were the most potent substrates.

Structure and function of Caulobacter crescentus aldose-aldose oxidoreductase.

Aldose-aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys(104) and Tyr(189), which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated.

A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

Swollenin from Trichoderma reesei exhibits hydrolytic activity against cellulosic substrates with features of both endoglucanases and cellobiohydrolases.

The cellulolytic and hemicellulolytic enzymes of Trichoderma reesei comprise one of the best characterised enzyme systems involved in lignocellulose degradation. In this paper, swollenin (SWOI), a protein recognised based on its sequence similarity with plant expansins, has been characterised. SWOI and its catalytic domain were subjected to analysis of their hydrolytic activity on different soluble carbohydrate polymers. By measuring the production of reducing ends, zymogram-, and viscosity analysis, SWOI was shown to have activity on substrates containing ß-1,4 glucosidic bonds, i.e. carboxymethyl cellulose, hydroxyethyl cellulose and ß-glucan. The formation of oligosaccharides from ß-glucan was analysed by HPLC and showed cellobiose as the main reaction product. SWOI was also able to hydrolyse soluble cello-oligosaccharides and the products formed were all consistent with SWOI cleaving a cellobiose unit off the substrate. In conclusion, the T. reesei swollenin showed a unique mode of action with similarities with action of both endoglucanases and cellobiohydrolases.

Structure and function of a decarboxylating Agrobacterium tumefaciens keto-deoxy-d-galactarate dehydratase.

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast.