Arbeitsgruppe Bioverfahrenstechnik

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Investigation of vitamin D2 and vitamin D3 hydroxylation by Kutzneria albida
(2021-03-01) Schmitz, Lisa Marie; Kinner, Alina; Althoff, Kirsten; Rosenthal, Katrin; Lütz, Stephan
The active vitamin D metabolites 25-OH−D and 1α,25-(OH)2−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2) and vitamin D3 (VD3) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH−D2 and 25-OH−D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH−D3 and tenfold higher concentrations of 25-OH−D2 after addition of 5 % (w/v) 2-hydroxypropyl β-cyclodextrin (2-HPβCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)2−D2 and 1,25-(OH)2−D3 and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites.
Is gender equality still an issue? Gender (im)balances in STEM
(2021-05-05) Rosenthal, Katrin
In recent years, considerable efforts have been made to support women to study and work in STEM fields. Nevertheless, inequalities remain, and women are clearly underrepresented in STEM. There is still a pervasive issue with the retention and progression of women in academia. How big is this problem actually, how can we promote diversity, equality and inclusion in academia, and how can we counteract unconscious bias?
Biotechnological production of cyclic dinucleotides - challenges and opportunities
(2021-12-25) Bartsch, Tabea; Becker, Martin; Rolf, Jascha; Rosenthal, Katrin; Lütz, Stephan
Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in prokaryotic and eukaryotic cells. As strong agonists of the stimulator of interferon genes, they are of great interest for pharmaceutical applications. In particular, cyclic-GMP-AMP and related synthetic CDNs are promising candidates in preclinical work and even some in clinical phase 1 and 2 studies. The comparison of chemical and biocatalytic synthesis routes elucidated that biological CDN synthesis offers some advantages, such as shorter synthesis time, avoiding complex protective group chemistry, and the access to a new spectrum of CDNs. However, the synthesis of CDNs in preparative quantities is still a challenge, since the chemical synthesis of CDNs suffers from low yields and complex synthetic routes and the enzymatically catalyzed synthesis is limited by low product titers and process stability. We aim to review the latest discoveries and recent trends in chemical and biocatalytic synthesis of CDNs with a focus on the synthesis of a huge variety of CDN derivatives. We furthermore consider the most promising biotechnological processes for CDN production by evaluating key figures of the currently known processes.
Catalytic promiscuity of cGAS: a facile enzymatic synthesis of 2′-3′-Linked cyclic dinucleotides
(2020-07-07) Rosenthal, Katrin; Becker, Martin; Rolf, Jascha; Siedentop, Regine; Hillen, Michael; Nett, Markus; Lütz, Stephan
Enzymatic shortcut: Cyclic dinucleotides, which are of great interest to study immunology and immune oncology, can be synthesized in a one-step biotransformation significantly shortening the chemical synthesis route. The enzyme displays a surprisingly large substrate scope. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP-AMP dinucleotide 2′3′-cGAMP. 2′3′-cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the α-thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives.
A gram-scale limonene production process with engineered escherichia coli
(2020-04-18) Rolf, Jascha; Julsing, Mattijs K.; Rosenthal, Katrin; Lütz, Stephan
Monoterpenes, such as the cyclic terpene limonene, are valuable and important natural products widely used in food, cosmetics, household chemicals, and pharmaceutical applications. The biotechnological production of limonene with microorganisms may complement traditional plant extraction methods. For this purpose, the bioprocess needs to be stable and ought to show high titers and space-time yields. In this study, a limonene production process was developed with metabolically engineered Escherichia coli at the bioreactor scale. Therefore, fed-batch fermentations in minimal medium and in the presence of a non-toxic organic phase were carried out with E. coli BL21 (DE3) pJBEI-6410 harboring the optimized genes for the mevalonate pathway and the limonene synthase from Mentha spicata on a single plasmid. The feasibility of glycerol as the sole carbon source for cell growth and limonene synthesis was examined, and it was applied in an optimized fermentation setup. Titers on a gram-scale of up to 7.3 g·Lorg−1 (corresponding to 3.6 g·L−1 in the aqueous production phase) were achieved with industrially viable space-time yields of 0.15 g·L−1·h−1. These are the highest monoterpene concentrations obtained with a microorganism to date, and these findings provide the basis for the development of an economic and industrially relevant bioprocess.
Accessing the biocatalytic potential for C−H‐activation by targeted genome mining and screening
(2019-08-14) Schmitz, Lisa Marie; Schäper, Jonas; Rosenthal, Katrin; Lütz, Stephan
Cytochrome P450 monooxygenases (P450s) are ubiquitous hemeproteins that insert oxygen specifically into substrates leading to diverse chemical transformations. Utilizing their capabilities, microbial whole‐cell biocatalysts are applied in pharmaceutical and fine chemical industry to produce biomolecules and drug metabolites. In order to synthesize novel bioactive compounds there is a great demand to identify P450s with new reaction and substrate scope. In this study, genome mining and an activity screening were successfully combined to discover so far underutilized biocatalysts. The screening revealed the expected broad range of reactions, such as hydroxylations, dealkylations, reductions and desaturations. For Actinosynnema mirum and ritonavir the biotransformation was transferred to a preparative scale resulting in a ritonavir conversion of 90 % after 48 h and 13 different metabolites analyzed by LC‐MS2 and NMR. These results clearly demonstrate the potential of the underlying approach to identify promising whole cell biocatalysts with good conversion and product scopes.
Recent advances in heme biocatalysis engineering
(2019-09-04) Schmitz, Lisa Marie; Rosenthal, Katrin; Lütz, Stephan
Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time‐limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine‐ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L−1. This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.
Screening and identification of novel cGAS homologues using a combination of in vitro and in vivo protein synthesis
(2019-12-22) Rolf, Jascha; Siedentop, Regine; Lütz, Stephan; Rosenthal, Katrin
The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.
Enhanced production and in situ product recovery of fusicocca-2,10(14)-diene from yeast
(2018-08-17) Halka, Lisa; Wichmann, Rolf
Fusicocca-2,10(14)-diene (FCdiene) is a tricyclic diterpene which has many pharmaceutical applications, for example, it is a precursor for different anticancer drugs, including fusicoccin A. Chemical synthesis of this diterpene is not economical as it requires 14 steps with several stereospecific reactions. FCdiene is naturally produced at low titers in phytopathogenic filamentous fungi. However, production of FCdiene can be achieved via expression of fusicoccadiene synthase in yeast. The objective of this study is to increase FCdiene production by optimizing the yeast fermentation process. Our preliminary fermentations showed influences of carbon sources, buffer agents, and oxygen supply on FCdiene production. Buffer agents as well as oxygen supply were investigated in detail at 0.2 and 1.8 L cultivation volumes. Using glucose as the carbon source, FCdiene concentrations were increased to 240 mgFCdiene/L by optimizing pH and oxygen conditions. In situ extraction and adsorption techniques were examined at the 0.2 L scale to determine if these techniques could improve FCdiene yields. Different adsorbents and solvents were tested with in situ product recovery and 4-fold increases in FCdiene productivity could be shown. The results generated in this work provide a proof-of-concept for the fermentative production of FCdiene from S. cerevisiae as a practical alternative to chemical synthesis.
An inert continuous microreactor for the isolation and analysis of a single microbial cell
(2015-11-30) Rosenthal, Katrin; Falke, Floris; Frick, Oliver; Dusny, Christian; Schmid, Andreas
Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level.
Production of rhamnolipids by integrated foam adsorption in a bioreactor system
(2018-07-24) Anic, Iva; Apolonia, Ines; Franco, Pedro; Wichmann, Rolf
Biosurfactants offer environmental as well as health benefits over traditionally used chemical surfactants and heterologous production from engineered microorganisms has been demonstrated, offering containable as well as scalable production of these alternative chemicals. Low product titers and cost intensive downstream processing are the main hurdles for economical biosurfactant production at industrial scales. Increased biosurfactant concentrations are found in the liquid fraction of the foam formed during fermentation of producing microbes. Adsorption of biosurfactants from foam fractions in cultivations may offer a simple concentration and purification method which could enable their cost-effective production. Here, foam adsorption was applied as an in situ method for separation of the rhamnolipid biosurfactants during fermentation of Pseudomonas putida EM383. An integrated process was designed to capture the produced rhamnolipids on hydrophobic adsorbent in packed bed units while minimizing the impact of adsorption on the productivity of the system by recirculating cell-containing collapsed foam flow-through back into the reactor vessel. A stable rhamnolipid production by P. putida EM383 on glucose was performed coupled to this adsorption strategy for 82 h, after which no remaining rhamnolipids were found in the cultivation broth and 15.5 g of rhamnolipids could be eluted from the adsorbent. Rhamnolipid yield from glucose feed was 0.05 g g−1, when up to 2 g L−1 glucose pulse feeding was applied. After solvent evaporation, a product purity of 96% was obtained. The results indicate that the integrated adsorption method can be efficient for simultaneous production and recovery of rhamnolipid biosurfactants from microbial fermentations.
Application of cell-free protein synthesis for faster biocatalyst development
(2019-02-19) Rolf, Jascha; Rosenthal, Katrin; Lütz, Stephan
Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

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