Cell envelopes for multi-enzyme synthesis: approaches for increasing the catalytic activity

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Microorganisms with overexpressed enzymes are frequently employed in biocatalytic reactions. These whole-cell biotransformations are attractive because they do not require expensive enzyme purification procedures. Disadvantages are, however, side reactions catalyzed by endogeneous enzymes and mass transfer limitations caused by the cell wall. These disadvantages can be avoided by using isolated enzymes, but they usually have to be immobilized to make processes economically viable. A a novel one-step expression and immobilization method on the basis of cellular envelopes made from bacteria combines key benefits of using whole cells and isolated enzymes. The enzymes catalyzing the desired reaction are genetically fused to membrane anchors which facilitate the immobilization of the expressed proteins in the cytoplasmic membrane of E. coli cells. Subsequently, a phage protein is expressed, which results in the formation of a lysis pore. The difference in osmotic pressure between the interior of the cell and the surrounding medium leads to the release of the cytoplasm through the formed pore, whereas immobilized enzymes are retained within the empty cellular envelopes. Amongst others it has been shown that cellular envelopes with immobilized beta-galactosidase clearly outperform the corresponding whole cells with overexpressed enzymes because the lysis pore reduces the mass transfer limitation of the substrate, which leads to a 3-fold higher enzyme activity.So far, only the cytoplasmic membrane of the cellular envelopes was used for enzyme immobilization. To increase the activity of the biocatalytic preparations, two strategies should be followed: On the one hand, enzymes should be additionally immobilized on the cellular surface using surface display. On the other hand, the inner space of the cellular envelopes should be utilized. For this purpose, structures have to be used that are not expelled from the cells during the lysis. Possible candidates are intracellular membranes, bacterial surface-layer proteins and bacterial cytoskeletons. As reaction system, a two-enzyme system consisting of an ene reductase from cyanobacteria and a cofactor-regenerating enzyme (formate dehydrogenase or glucose dehydrogenase) should be investigated, which will be used for the reduction of (R)-carvone to (2R,5R)-dihydrocarvone. A reaction engineering analysis of asymmetric syntheses using the different cellular envelopes with immobilized enzymes should be made. The cellular envelopes with maximum enzyme activities, which should also be as balanced as possible, are to be compared with whole cells and isolated enzymes in biotransformations performed in one- and two-phase systems. Finally, the scalability of the production of cellular envelopes and their work-up using tangential flow filtration as well as of the asymmetric synthesis should be investigated.