Abstract:Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole… Show more
“…It is known that the reaction is mediated by a tryptophanase (TnaA) in E. coli and can be avoided by introducing a deletion of the tnaA gene. Using a tnaA deficient strain like BW25113 ∆ tnaA , that is available in the Keio collection, could be a solution to address the cross contamination of the product [ 22 , 29 , 30 , 31 ]. Fig 4 Biotransformations were upscaled to semi-preparative reaction volume with the substrate naphthalene.…”
Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of
cis
-dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology,
e.g.
chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of
Pseudomonas fluorescens
IP01 and its variant CDO M232A expressed in
E. coli
JM109 (DE3) cells, employing
R
-limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale.
Preservation of biocatalysts in recombinant whole-cells.
Ready-to-use enzymatic reaction.
Semi-preparative biotransformation with lyophilized whole-cells.
“…It is known that the reaction is mediated by a tryptophanase (TnaA) in E. coli and can be avoided by introducing a deletion of the tnaA gene. Using a tnaA deficient strain like BW25113 ∆ tnaA , that is available in the Keio collection, could be a solution to address the cross contamination of the product [ 22 , 29 , 30 , 31 ]. Fig 4 Biotransformations were upscaled to semi-preparative reaction volume with the substrate naphthalene.…”
Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of
cis
-dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology,
e.g.
chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of
Pseudomonas fluorescens
IP01 and its variant CDO M232A expressed in
E. coli
JM109 (DE3) cells, employing
R
-limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale.
Preservation of biocatalysts in recombinant whole-cells.
Ready-to-use enzymatic reaction.
Semi-preparative biotransformation with lyophilized whole-cells.
“…26 To facilitate screening with E. coli, a tryptophanase-deficient cell strain was used, E. coli DE3 (C43) ΔTnaA, previously engineered in the Bernardt group. 27 The removal of TnaA was shown not to affect the growth rate of E. coli and was, therefore, well suited for directed evolution experiments. 27 First, we probed the Trp biosensor in a microtiter plate-based assay.…”
Section: Enrichment Of Active Trpb In Plate-based and Droplet-based F...mentioning
confidence: 99%
“…27 The removal of TnaA was shown not to affect the growth rate of E. coli and was, therefore, well suited for directed evolution experiments. 27 First, we probed the Trp biosensor in a microtiter plate-based assay. TrpB 7E6 and negative control (plasmid without insert) were transformed and expressed in a 1 mL culture of E. coli DE3 (C43) ΔTnaA.…”
Section: Enrichment Of Active Trpb In Plate-based and Droplet-based F...mentioning
Tryptophan synthase catalyzes the synthesis of a wide array of non-canonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (>107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic read-outs which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a biosensor to quantitatively report on tryptophan production in droplets. The utility of the biosensor was validated by identifying 5-fold improved tryptophan synthases from ~100,000 protein variants. More generally this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.
“…The improvement was brought about by merely adding one more mutation. Moreover, in the last two decades, more microbial CYPs have been reported for their capacity of indigo formation (Brixius-Anderko et al 2017;Fiorentini et al 2018;Kim et al 2018) which may also represent good starting points for creating an optimized CYP for indigo production. The feature that conversion of indole into indigo can be seen by eye makes it attractive to apply directed evolution protocols.…”
Indigo is one of the oldest textile dyes and was originally prepared from plant material. Nowadays, indigo is chemically synthesized at a large scale to satisfy the demand for dyeing jeans. The current indigo production processes are based on fossil feedstocks; therefore, it is highly attractive to develop a more sustainable and environmentally friendly biotechnological process for the production of this popular dye. In the past decades, a number of natural and engineered enzymes have been identified that can be used for the synthesis of indigo. This mini-review provides an overview of the various microbial enzymes which are able to produce indigo and discusses the advantages and disadvantages of each biocatalytic system.
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