Vera D. Jäger

Selective Enzymatic Synthesis of the Grapefruit Flavor (+)‐Nootkatone

(+)‐Nootkatone is a high‐value sesquiterpenoid known for its grapefruit‐odor impression. Its isolation from natural plant sources suffers from low yields, and chemical syntheses involve carcinogenic or hazardous compounds. Herein, a biocatalytic route for the synthesis of (+)‐nootkatone that combines two enzymes in one pot is presented. In the first step, a cytochrome P450 monooxygenase catalyzes the selective allylic hydroxylation of the sesquiterpene (+)‐valencene to the intermediate alcohol nootkatol. In the second step, nootkatol is further oxidized to (+)‐nootkatone by an alcohol dehydrogenase (ADH). The challenging task of finding a suitable cofactor regeneration system was solved by careful selection of an appropriate cosubstrate for the ADH, which works in a dual‐functional mode. After reaction optimization, involving cosolvent and cosubstrate screening, (+)‐nootkatone concentrations of up to 360u2005mgu2009L−1 and a space‐time yield of 18u2005mgu2009L−1u2009h−1 were achieved.

Catalytically-active inclusion bodies—Carrier-free protein immobilizates for application in biotechnology and biomedicine

Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation.

Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bodies (CatIBs) of the constitutive L-lysine decarboxylase from E. coli (EcLDCc-CatIBs) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum. EcLDCc-CatIBs can easily be produced in E. coli followed by a simple purification protocol yielding up to 43% dry CatIBs per dry cell weight. The stability and recyclability of EcLDCc-CatIBs was demonstrated in (repetitive) batch experiments starting from L-lysine concentrations of 0.1u2009M and 1u2009M. EcLDC-CatIBs exhibited great stability under reaction conditions with an estimated half-life of about 54u2009h. High conversions to DAP of 87–100% were obtained in 30–60u2009ml batch reactions using approx. 180–300u2009mg EcLDCc-CatIBs, respectively. This resulted in DAP titres of up to 88.4u2009gu2009l−1 and space-time yields of up to 660 gDAP l−1 d−1 per gram dry EcLDCc-CatIBs. The new process for DAP production can therefore compete with the currently best fermentative process as described in the literature.

A Synthetic Reaction Cascade Implemented by Colocalization of Two Proteins within Catalytically Active Inclusion Bodies

In nature, enzymatic reaction cascades, i.e., realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of learning from nature has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial cascades. Hereby, the spatial organization of multiple enzymes, e.g., by coimmobilization, remains a challenging task, as currently no generic principles are available that work for every enzyme. We here present a tunable, genetically programmed coimmobilization strategy that relies on the fusion of a coiled-coil domain as aggregation inducing-tag, resulting in the formation of catalytically active inclusion body coimmobilizates (Co-CatIBs). Coexpression and coimmobilization was proven using two fluorescent proteins, and the strategy was subsequently extended to two enzymes, which enabled the realization of an integrated enzymatic two-step cascade for the production of (1 R,2 R)-1-phenylpropane-1,2-diol (PPD), a precursor of the calicum channel blocker diltiazem. In particular, the easy production and preparation of Co-CatIBs, readily yielding a biologically produced enzyme immobilizate renders the here presented strategy an interesting alternative to existing cascade immobilization techniques.

Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.