Konrad B. Otte

Enzyme engineering in the context of novel pathways and products.

In recent years, enzyme engineering was used to fine-tune a diverse set of proteins to realize new biosynthetic pathways and gain access to novel products. However, enzymes in nature do not always meet the required demands in terms of activity, selectivity and stability. In these cases enzyme engineering has been used to improve the enzyme properties, which facilitated the development of tailor-made functional biocatalysts, even beyond their natural capabilities. Examples can be found in the three main areas of chemical biotechnology: single-step biocatalysis, metabolic engineering and enzymatic cascades. In this review we highlight recently published work in all of these three fields and emphasize the main trends and differences.

Synthesis of 9‐Oxononanoic Acid, a Precursor for Biopolymers

Polymers based on renewable resources have become increasingly important. The natural functionalization of fats and oils enables an easy access to interesting monomeric building blocks, which in turn transform the derivative biopolymers into high-performance materials. Unfortunately, interesting building blocks of medium-chain length are difficult to obtain by traditional chemical means. Herein, a biotechnological pathway is established that could provide an environmentally suitable and sustainable alternative. A multiple enzyme two-step one-pot process efficiently catalyzed by a coupled 9S-lipoxygenase (St-LOX1, Solanum tuberosum) and 9/13-hydroperoxide lyase (Cm-9/13HPL, Cucumis melo) cascade reaction is proposed as a potential route for the conversion of linoleic acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to give 9S-hydroperoxy-octadecadienoic acid (9S-HPODE) as a cascade intermediate, which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot process afforded a yield of 73 % combined with high selectivity. The best reaction performance was achieved when lipoxygenase and hydroperoxide lyase were applied in a successive rather than a simultaneous manner. Green leaf volatiles, which are desired flavor and fragrance products, are formed as by-products in this reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13-HPLs, which exhibited a strong preference for 9S-HPODE over 9R-HPODE.

Whole-Cell One-Pot Biosynthesis of Azelaic Acid

Polymers benefit from the use of biogenic resources such as fatty acids. They enable easy access to valuable monomeric building blocks, which, in comparison to their exclusively fossil counterparts, lead to products with improved physicochemical properties. Monomers of special interest are medium‐chain dicarboxylic acids, which are not easy to obtain by traditional chemical means. Previously, we established an in vitro pathway that combined a 9‐lipoxygenase and a 9/13‐hydroperoxide lyase, which enabled the conversion of linoleic acid via a hydroperoxy intermediate into 9‐oxononanoic acid, the precursor of azelaic acid. Herein, we aimed for the further development of the multi‐enzyme cascade, which included the oxidation of 9‐oxononanoic acid and the establishment of a suitable whole‐cell catalyst. A detailed investigation of the simultaneous in vitro reaction setup revealed that both lipoxygenase activation and the subsequent hydroperoxide lyase reaction depend on the hydroperoxide reaction intermediate. For the activation of lipoxygenase, the hydroperoxide lyase activity, therefore, has to be significantly reduced. In accordance with these observations, we established a suitable dual‐expression system and we further demonstrated that endogenous E. coli redox enzymes are feasible to oxidize 9‐oxononanoic acid to azelaic acid. The resulting whole‐cell catalyst is, therefore, able to perform the direct bioconversion of linoleic acid into azelaic acid. The use of organic solvent as the second phase improved the overall performance of the E. coli host strain. The developed one‐pot, single‐step process afforded 29 mg L−1 of azelaic acid within 8 h with a substrate conversion of 34 % and a selectivity of 47 %.

Synthesis of Sebacic Acid Using a De Novo Designed Retro-Aldolase as a Key Catalyst

The production of new organic compounds from natural feedstocks drives the construction of new biosynthetic cascades. To develop and generate valuable complex chemicals, it is necessary to go beyond natural enzymes towards de novo designed and engineered variants for both natural and non‐natural compounds. Herein, we exploit the applicability of a de novo retro‐aldolase for the synthesis of sebacic acid, a valuable precursor for bio‐based polymers. From ricinoleic acid, a route composed of four individual enzyme‐catalyzed steps was developed. As a result of the narrow substrate scope of natural aldolases, this enzymatic route could only be realized by the design and engineering of a de novo retro‐aldolase. We further highlight the outstanding potential of this technology for the realization of non‐natural reactions and pathways.

Optimized Reaction Conditions Enable the Hydration of Non-natural Substrates by the Oleate Hydratase from Elizabethkingia meningoseptica

The oleate hydratase from Elizabethkingia meningoseptica (Em‐OAH) catalyzes the hydration of oleic acid (C18) to (R)‐10‐hydroxystearic acid. In previous work, low activity of Em‐OAH towards chemically synthesized (Z)‐undec‐9‐enoic acid (C11) was observed. Product formation in the hydration of the truncated C11 substrate was improved by optimizing the reaction conditions by applying statistical experiment design. Optimized reaction conditions resulted in a 2.8‐fold increase in product formation in just one quarter of the time (64 % conversion in 28 h). The applicability has been assessed in the upscaling of the conversion of (Z)‐undec‐9‐enoic acid to (S)‐10‐hydroxyundecanoic acid (132 mg product, >95 % purity). Reaction conditions developed for the hydration of C11 facilitated the first hydration of non‐natural alkenes. By using a fatty acid dummy substrate, 1‐decene was successfully hydrated to (S)‐2‐decanol with excellent stereoselectivity and 50 % conversion after four days of incubation.

Production of ω-hydroxy octanoic acid with Escherichia coli.

The present proof-of-concept study reports the construction of a whole-cell biocatalyst for the de novo production of ω-hydroxy octanoic acid. This was achieved by hijacking the natural fatty acid cycle and subsequent hydroxylation using a specific monooxygenase without the need for the additional feed of alkene-like precursors. For this, we used the model organism Escherichia coli and increased primarily the release of the octanoic acid precursors by overexpressing the plant thioesterase FatB2 from Cuphea hookeriana in a β-oxidation deficient strain, which lead to the production of 2.32mM (8.38mggcww(-1)) octanoic acid in 24h. In order to produce the corresponding ω-hydroxy derivative, we additionally expressed the engineered self-sufficient monooxygenase fusion protein CYP153AMaq(G307A)-CPRBM3 within the octanoic acid producing strain. With this, we finally produced 234μM (0.95mggcww(-1)) ω-hydroxy octanoic acid in a 20h fed-batch set-up.