A combined experimental and modelling approach for the Weimberg pathway optimisation.

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.

Chemoenzymatic Cascade Synthesis of Optically Pure Alkanoic Acids by Using Engineered Arylmalonate Decarboxylase Variants.

Arylmalonate decarboxylase (AMDase) catalyzes the cofactor-free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short-chain 2-methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R- and S-selective AMDase variants produced optically pure short-chain 2-methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee).

Cloning and characterization of a new delta-specific l-leucine dioxygenase from Anabaena variabilis.

Optically pure hydroxy amino acids show several bioactivities and are valuable building blocks for the pharmaceutical industry. Fe(II)/α-ketoglutarate dependent dioxygenases catalyze the hydroxylation or sulfoxidation of l-amino acids with high regio- and stereoselectivity. While several ß- and γ-specific enzymes have been described, only one δ-specific hydroxylase has been reported so far. Based on its similarity to the known l-leucine 5-hydroxylase from Nostoc punctiforme, an open reading frame from the cyanobacterium Anabaena variabilis was identified as putative l-leucine dioxygenase (AvLDO). Here we report the cloning and characterization of this dioxygenase. The enzyme showed a high preference for acidic conditions and moderate reaction temperatures. AvLDO catalyzed the regio- and stereoselective hydroxylation of several aliphatic amino acids in δ-position. In case of the sulfoxidation of l-methionine, AvLDO produced the opposite diastereomer than isoleucine dioxygenase. AvLDO is thus an interesting addition to the toolbox of Fe(II)/α-ketoglutarate dependent dioxygenases. On the genomic DNA of Anabaena variabilis ATCC 29413, the avldo gene is located on a gene cluster involved (2S,4S)-4-methylproline biosynthesis, which is contained in bioactive peptides often found from cyanobacteria. This fact suggests the metabolic functional role of this amino acid dioxygenase in cyanobacteria.

Improvement of the Process Stability of Arylmalonate Decarboxylase by Immobilization for Biocatalytic Profen Synthesis.

The enzyme arylmalonate decarboxylase (AMDase) enables the selective synthesis of enantiopure (S)-arylpropinates in a simple single-step decarboxylation of dicarboxylic acid precursors. However, the poor enzyme stability with a half-life time of about 1.2 h under process conditions is a serious limitation of the productivity, which results in a need for high catalyst loads. By immobilization on an amino C2 acrylate carrier the operational stability of the (S)-selective AMDase variant G74C/M159L/C188G/V43I/A125P/V156L was increased to a half-life of about 8.6 days, which represents a 158-fold improvement. Further optimization was achieved by simple immobilization of the cell lysate to eliminate the cost- and time intensive enzyme purification step.

A Multi-Enzymatic Cascade Reaction for the Stereoselective Production of γ-Oxyfunctionalyzed Amino Acids.

A stereoselective three-enzyme cascade for synthesis of diasteromerically pure γ-oxyfunctionalized α-amino acids was developed. By coupling a dynamic kinetic resolution (DKR) using an N-acylamino acid racemase (NAAAR) and an L-selective aminoacylase from Geobacillus thermoglucosidasius with a stereoselective isoleucine dioxygenase from Bacillus thuringiensis, diastereomerically pure oxidized amino acids were produced from racemic N-acetylamino acids. The three enzymes differed in their optimal temperature and pH-spectra. Their different metal cofactor dependencies led to inhibitory effects. Under optimized conditions, racemic N-acetylmethionine was quantitatively converted into L-methionine-(S)-sulfoxide with 97% yield and 95% de. The combination of these three different biocatalysts allowed the direct synthesis of diastereopure oxyfunctionalized amino acids from inexpensive racemic starting material.

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance.

A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions. In this study, simultaneous saturation mutagenesis with a restricted set of amino acids at each position was applied to further heighten the activity of the (S)-selective AMDase variant toward α-methyl-α-phenylmalonate. The best variant (V43I/G74C/A125P/V156L/M159L/C188G) showed 9,500-fold greater catalytic efficiency kcat/Km than that of G74C/C188S. Notably, a high level of decarboxylation of α-(4-isobutylphenyl)-α-methylmalonate by the sextuple variant produced optically pure (S)-ibuprofen, an analgesic compound which showed 2.5-fold greater activity than the (R)-selective wild-type AMDase.

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C.

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Draft Genome Sequence of Bordetella bronchiseptica KU1201, the First Isolation Source of Arylmalonate Decarboxylase.

The analysis of the 6.8-Mbp draft genome sequence of the phenylmalonate-assimilating bacterium Bordetella bronchiseptica KU1201 identified 6,358 protein-coding sequences. This will give us an insight into the catabolic variability of this strain for aromatic compounds, along with the roles of arylmalonate decarboxylases in nature.