Protein engineering and synthetic application of the arylmalonate decarboxylase from Bordetella bronchiseptica\textit {Bordetella bronchiseptica}

Die Aktivität der enantioselektiven Arylmalonat-Decarboxylase (AMDase), wurde mittels eines kombinatorischen Ansatzes aus gerichteter Evolution und rationalem Protein Design für die Synthese von Profenen (Antirheumatika) gesteigert. Zur Aufklärung der Substratbindung wurden ATR-FTIR- und STD-NMR-spektroskopische Untersuchungen durchgeführt. Eine Optimierung der Proteinexpression und der $\textit {proof of concept}$ der photoautotrophen Expression in $\it Synechocystis$ verbesserten die Bereitstellung des Biokatalysators. Die Etablierung einer neuen Syntheseroute, zur Herstellung der Arylmalonsäurederivate, mit geänderter Schutzgruppenstrategie, führte, in Verknüpfung mit den verbesserten AMDase-Varianten, zur erfolgreichen Synthese von optisch reinem ($\it R$)-Naproxen sowie ($\it R$)- und ($\it S$)-Flurbiprofen. Die Kombination aus verbesserter Synthese und optimierten Reaktionsbedingungen ergab für instabile Arylmalonsäurederivate wie Flurbiprofenmalonsäure, eine deutliche ee-Steigerung des Produktes von 92 % auf 99 %

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

Photosynthetic production of enantioselective biocatalysts

$\bf Background:$ Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium $\it Synechocystis$ sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes. $\bf Results:$ We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in $\textit {E. coli}$ yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism $\textit {Sulfolobus tokodai}$ ST0071 and arylmalonate decarboxylase from $\textit {Bordetella bronchiseptica}$ showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates. $\bf Conclusions:$ We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from $\it Synechocystis$ did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels