Engineering decarboxylases for consolidated bioprocessing and more

Decarboxylases have been widely applied for the production of chemical building blocks from biomass derivatives as well as chiral intermediates in organic synthesis. First we have engineered different enzymes of this class (KDCA and PDC) for highly increased thermostability by combining rational design and random approaches. With melting points increased by almost 15 °C and half lifes above 70 °C prolonged up to several thousand fold this now opens up new possibilities for the production of alcohols such as ethanol, butanol or isobutanol from lignocellulose using thermophilic organisms in so called “consolidated bioprocessing” approaches, where lingo cellulose break-up is done simultaneously with product formation. In addition variants for higher tolerance towards denaturing, partially water miscible organic solvents were developed to be utilized e.g. for organic synthesis or in cell-free cascades like for the production of isobutanol from sugar. Often, decarboxylases show broad substrate promiscuity, which can be of advantage but also a major challenge in fine chemical production. By further engineering the substrate specificity and modifying the promiscuity, it was possible to modulate the formation of desired chemicals with high selectivity. Based on this we have designed a network of artificial enzymatic cascade reactions towards production of bio-based building blocks utilizing decarboxylases as the modulating enzymes. With engineered variants, we demonstrate the conversion of industrial byproducts into several fine chemicals with high selectivity and yield. This result also sheds light on substrate recognition of the class of decarboxylases towards more-challenging substrates, e.g. bulkier side chains for broader applicability

Oxidative biocatalysis without oxygen – Applying the less used side of hydrogenases

Please click Additional Files below to see the full abstract

A non-natural Nicotinamide cofactor for biotransformation at extreme conditions

Redox enzymes are very useful tools for establishing greener routes in organic synthesis, mostly due to their highly selective reduction, oxidation and oxyfunctionalisation reactions. However, the metastable nicotinamide cofactors (NAD/P(H)), required for catalysis, are prone to fast degradation, when utilized under non physiological conditions. Establishing of robust artificial enzymatic cascade reactions and cell free biotransformations at extreme conditions is therefore still a major challenge. We show one NAD(P)+ derivative with highly increased stability and very similar redox potential compared to its natural counterpart that can be used very efficiently with a number of biocatalysts. We have tested more than 50 redox enzymes and found a substantial number of them being able to utilize the nicotinamide derivative. Among them, we also successfully identified those that are commonly used in cofactor regeneration systems. Applying enzyme engineering to a model enzyme, it was possible to develop variants with activities even higher than towards the natural cofactor, allowing efficient biotransformations at 60°C and above for many hours. In addition further characterization and additional comparative molecular dynamic simulations revealed an improved understanding of the biomimick’s recognition on a molecular level, facilitating the transfer to other enzymes

In-depth rheological characterization of genetically modified xanthan-variants

Xanthan is an extensively studied viscosifying agent discovered in 1961. Acetylation and pyruvylation have a major influence on its rheological properties and the effect of these groups on the conformation and rheological properties of xanthan have been studied for decades. However, these studies rely mainly on chemical modifications and therefore the degree of pyruvylation and acetylation as well as regioselectivity of deacetylation cannot be controlled. Here, we present an in-depth rheological characterization of natural xanthan and seven xanthan-variants, with defined acetylation and pyruvylation patterns created via genetic modification of Xanthomonas campestris LMG 8031. By that approach xanthan-variants with defined acetylation and pyruvylation patterns in their most natural state due to the mild production conditions were obtained. It was possible to link the defined substituent patterns to their corresponding rheological properties to give novel structure-function relationship insights of xanthan-variants in salt-free environments and in the presence of mono- and divalent cations

Activated carbon as catalyst support: precursors, preparation, modification and characterization

The preparation of activated carbon materials is discussed along selected examples of precursor materials, of available production and modification methods and possible characterization techniques. We evaluate the preparation methods for activated carbon materials with respect to its use as catalyst support and identify important parameters for metal loading. The considered carbon sources include coal, wood, agricultural wastes or biomass as well as ionic liquids, deep eutectic solvents or precursor solutions. The preparation of the activated carbon usually involves pre-treatment steps followed by physical or chemical activation and application dependent modification. In addition, highly porous materials can also be produced by salt templating or ultrasonic spray pyrolysis as well as by microwave irradiation. The resulting activated carbon materials are characterized by a variety of techniques such as SEM, FTIR, nitrogen adsorption, Boehm titrations, adsorption of phenol, methylene blue and iodine, TPD, CHNS/O elemental analysis, EDX, XPS, XRD and TGA

Molecular dynamics provides insights into an engineered oxidoreductase with altered cofactor specificity

The aldehyde dehydrogenase from Thermoplasma acidophilum is one of the key enzymes in a previously established synthetic cell-free reaction cascade for the production of alcohols. In order alter the cofactor specificity of this enzyme from NADP+ to NAD+, we applied the CSR-salad tool and investigated further amino acid positions based on its crystal structure. Introduction of five point mutations reduced the Km for NAD+ from 18 mM to 0.6 mM and simultaneously increased the activity for D-glyceraldehyde from 0.4 U/mg to 1.5 U/mg. In order to understand the structural basis of the beneficial mutations, we performed molecular dynamics simulations that showed a significant flexibility gain at the cofactor binding site of the final variant. This increased flexibility facilitates a loop movement that largely contributes to the gain in activity and cofactor specificity. We envision a future optimization potential for aldehyde dehydrogenases based on our results

The correlation between NAD(P)H oxidase kinetics and its stability exposed to gas- liquid interface

Please click Additional Files below to see the full abstrac

Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii

Schmid J, Müller-Hagen D, Bekel T, et al. Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii. BMC Genomics. 2010;11(1): 329.Background The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 -> 3)-[beta]-linked glucose with a (1 -> 6)-(beta)-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches. Results Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields. Conclusions The results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to identify important genes putatively involved in determining scleroglucan yields. Moreover, our data establish the first sequence database for S. rolfsii, which allows research into other biological processes of S. rolfsii, such as host-pathogen interaction