Photosynthetic production of enantioselective biocatalysts

Abstract

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 Synechocystis sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes.

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 E. coli yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism Sulfolobus tokodai ST0071 and arylmalonate decarboxylase from Bordetella bronchiseptica showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates.

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 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.

Figure 1

Cyanobacterial enzyme expression of enantioselective enzymes is a sustainable source of biocatalysts for the chemical industry.

Figure 2

Plasmids for the site-directed genome integration of genes by homologous recombination.

Figure 3

Confirmation of successful genome integration of a) the ST0071 gene and b) the AMDase gene by PCR.

Figure 4

Cultivation in 5 L scale of Synechocystis wild type and recombinant strains with protein yields after 96 h.

Figure 5

Functional expression of esterase ST0071 in Synechocystis sp. 6803. (a) SDS-PAGE of a functional expression of the sfGFP-fusion protein of esterase ST0071 (64 kD) in Synechocystis sp. PCC 6803 (b) Selective activity staining using α-naphthylacetate and Fast Red [29]. Cultures were grown in BG-11 media [28] in shake flasks and tubes at 30°C under a light strength of 300 μmol photons m^-2 s^-1 under an atmosphere containing 5% of CO₂.

Figure 6

Esterase-catalysed desymmetrization of a prochiral malonic acid ester. Cell lysate from a cultivation Synechocystis sp. sfGFP-ST0071 cell lysate (500 μL of a 750 mL cultivation, OD 6.5) was added to a solution of 5 mM phenyl methyl malonate diethyl ester 1 and incubated for 48 h at 37°C. The reaction was stopped by addition of HCl (200 μL, 2 M), extracted twice with methyl tert-butyl ester. The optical purity of (S)-2 was determined using by chiral HPLC.

Figure 7

Arylmalonate decarboxylase-catalyzed desymmetrization of prochiral malonic acids. a) Desymmetrization of prochiral malonic acids 3a and 4b at 30°C using cell lysates from a cultivation of Synechocystis sp. sfGFP-AMDase. b) GC-chromatogram of optically pure (R)-4a (grey) and a racemic standard (light grey).