Autodisplay of glucose-6-phosphate dehydrogenase for redox cofactor regeneration at the cell surface.

Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface. The obtained whole cell biocatalyst demonstrated similar kinetic parameters compared to the purified enzyme, more precisely KM values of 0.2 mM for NADP+ and calculated total turnover numbers of 107 . However, the KM for the substrate G6P increased by a factor of 7 to yield 1.5 mM. The whole cell biocatalyst was cheaper to produce, easy to separate from the reaction mixture and reusable in consecutive reaction cycles. Furthermore, lyophilization allowed storage at room temperature. The whole cell biocatalyst displaying G6PDH was applicable for NADPH regeneration in combination with soluble as well as surface displayed enzymes and model reactions in combination with bacterial CYP102A1 and human CYP1A2 were realized. Biotechnol. Bioeng. 2017;114: 1658-1669. © 2017 Wiley Periodicals, Inc.

Improving the activity of surface displayed cytochrome P450 enzymes by optimizing the outer membrane linker.

Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded ß-barrel of the autotransporter. The linker can have influence on aspects such as steric orientation of the passenger, distance to the outer membrane and accessibility of active sites. Six linker variants for display of the cytochrome P450 reductase were tested. Cytochrome c reduction by the cytochrome P450 reductase varied fivefold and was highest by introduction of a flexible glycine-serine region. When these variants were co-expressed with surface displayed CYP1A2, product concentration for paracetamol differed between 0.22 µM and 2.5 µM and for resorufin between 0.23 µM to 1 µM. The best glycine/serine containing sequence, that turned out to be best for CPR display, was then introduced into the linker for displaying CYP1A2. In comparison, up to 7.9 µM paracetamol and up to 1.69 µM resorufin were obtained with this new variant. The differences were not caused by changes in the number of displayed enzymes. To our knowledge, this is the first systematic study on engineering the linker for surface display of recombinant enzymes.

Co-expression of active human cytochrome P450 1A2 and cytochrome P450 reductase on the cell surface of Escherichia coli.

BACKGROUND: Human cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug-drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli. RESULTS: To present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme. CONCLUSION: We demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.

Ethylene production in relation to nitrogen metabolism in Saccharomyces cerevisiae.

We have previously shown that ethylene production in Saccharomyces cerevisiae expressing the ethylene-forming enzyme (EFE) from Pseudomonas syringae is strongly influenced by variations in the mode of cultivation as well as the choice of nitrogen source. Here, we have studied the influence of nitrogen metabolism on the production of ethylene further. Using ammonium, glutamate, glutamate/arginine, and arginine as nitrogen sources, it was found that glutamate (with or without arginine) correlates with a high ethylene production, most likely linked to an observed increase in 2-oxoglutarate levels. Arginine as a sole nitrogen source caused a reduced ethylene production. A reduction of arginine levels, accomplished using an arginine auxotrophic ARG4-deletion strain in the presence of limiting amounts of arginine or through CAR1 overexpression, did however not correlate with an increased ethylene production. As expected, arginine was necessary for ethylene production as ethylene production in the ARG4-deletion strain ceased at the time when arginine was depleted. In conclusion, our data suggest that high levels of 2-oxoglutarate and a limited amount of arginine are required for successful ethylene production in yeast.

Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

Identification of factors for improved ethylene production via the ethylene forming enzyme in chemostat cultures of Saccharomyces cerevisiae.

BACKGROUND: Biotechnological production of the traditional petrochemical ethylene is presently being explored using yeasts as well as bacteria. In this study we quantify the specific ethylene production levels at different conditions in continuous (chemostat) cultivation of Saccharomyces cerevisae expressing the ethylene forming enzyme (EFE) from Pseudomonas syringae. RESULTS: Our study shows that oxygen availability is an important factor for the ethylene formation. Maintaining a high percentage dissolved oxygen in the cultivation was found to be necessary to achieve maximal ethylene productivity. Even at oxygen levels high enough to sustain respiratory metabolism the ethylene formation was restricted. Oxygen was also important for sustaining a high respiratory rate and to re-oxidize the surplus of NADH that accompanies ethylene formation. By employing three different nitrogen sources we further found that the nitrogen source available can both improve and impair the ethylene productivity. Contrary to findings in batch cultures, using glutamate did not give a significant increase in specific ethylene production levels compared to the reference condition with ammonia, whereas a combination of glutamate and arginine resulted in a strongly diminished specific ethylene production. Furthermore, from cultivations at different dilution rates the ethylene formation was found to be coupled to growth rate. CONCLUSION: To optimize the ethylene productivity in S. cerevisiae expressing a bacterial ethylene forming enzyme, controlling the oxygen availability and growth rate as well as employing an ideal nitrogen source is of importance. The effects of these factors as studied here provide a basis for an optimized process for ethylene production in S. cerevisiae.