Thomas Haas

Redox Self-Sufficient Biocatalyst Network for the Amination of Primary Alcohols†

Driving the machinery: A biocatalytic redox-neutral cascade for the preparation of terminal primary amines from primary alcohols at the expense of ammonia has been established in a one-pot one-step method. Applying this artificial biocatalyst network, long-chain 1,ω-alkanediols were converted into diamines, which are building blocks for polymers, in up to 99 % conversion.

Oxidative Decarboxylation of Short‐Chain Fatty Acids to 1‐Alkenes

The enzymatic oxidative decarboxylation of linear short-chain fatty acids (C4:0-C9:0) employing the P450 monooxygenase OleT, O2 as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C3 to C8  alkenes with product titers of up to 0.93 g L(-1) and TTNs of >2000. Key to this process was the construction of an efficient electron-transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1-alkenes, such as propene and 1-octene, from renewable resources for the first time.

Artificial Multi‐Enzyme Networks for the Asymmetric Amination of sec‐Alcohols

Various artificial network designs that involve biocatalysts were tested for the asymmetric amination of sec-alcohols to the corresponding α-chiral primary amines. The artificial systems tested involved three to five redox enzymes and were exemplary of a range of different sec-alcohol substrates. Alcohols were oxidised to the corresponding ketone by an alcohol dehydrogenase. The ketones were subsequently aminated by employing a ω-transaminase. Of special interest were redox-neutral designs in which the hydride abstracted in the oxidation step was reused in the amination step of the cascade. Under optimised conditions up to 91 % conversion of an alcohol to the amine was achieved.

Coupled Enzymatic Alcohol‐to‐Amine Conversion of Isosorbide using Engineered Transaminases and Dehydrogenases

A matching dehydrogenase and transaminase pair was engineered with regard to substrate recognition, catalytic activity, and cofactor specificity with the final goal to convert the bicyclic dialcohol isosorbide into a diamine by a multistep biocatalytic process. Individual catalytic turnover rates as well as coupled conversion to the amine were investigated for the enzymes in analytical assays that used the substrate isosorbide and different intermediates along the multiple pathway reaction cascade, in particular, stereoisomers of hydroxy ketones, amino alcohols, and amino ketones. For parallel screening and evaluation of mutant enzymes with regard to catalytic activities and optimal reaction conditions, a robotic platform was established that comprised all steps from bacterial protein expression to the enzymatic assay. As a result, we present a three‐enzyme system composed of L. aquatica levodione reductase, an engineered P. denitrificans ω‐aminotransferase, and B. subtilis alanine dehydrogenase that catalyzes formation of the isosorbide monoamine with a yield of up to ≈7 % under our analytical assay conditions. After further optimization and adaptation to whole‐cell catalysis, this enzyme system may open a biotechnologically attractive route to a structurally rigid diamine for the production of biosynthetic polymer materials.

Aerobic oxidation of isosorbide and isomannide employing TEMPO/laccase

The oxidation of the renewable diols isosorbide and isomannide was successfully achieved using a TEMPO/laccase system. Furthermore, various TEMPO-derivatives were tested leading to conversions of up to >99% for the oxidation of isosorbide, isomannide, indanol and a halohydrin to the corresponding ketone.

Biocatalytic Oxidative Cascade for the Conversion of Fatty Acids to alpha-Ketoacids via Internal H2O2 Recycling

Abstract The functionalization of bio‐based chemicals is essential to allow valorization of natural carbon sources. An atom‐efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α‐ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α‐hydroxylation of fatty acids combined with enantioselective oxidation by α‐hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H2O2, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O2‐dependent cascade relies on catalytic amounts of H2O2 and releases water as sole by‐product. Octanoic acid was converted under mild conditions in aqueous buffer to 2‐oxooctanoic acid in a simultaneous one‐pot two‐step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale‐up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

Biotechnologische Herstellung terminaler Alkene und Diene

Abstract1-Alkenes represent primary building blocks for the chemical industry. Here we show the development of a novel biosynthetic redox-cascade involving the P450 monooxygenase OleT and electron transfer proteins CamA and CamB to allow the biocatalytic synthesis of short chain alkenes such as propene or 1,4-pentadiene from fatty acids. The cascade is fully based on renewables and can be operated under mild reaction conditions as opposed to industrial alkene synthesis.