Is gender equality still an issue? Gender (im)balances in STEM

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

Agar plate-based screening approach for the identification of enzyme-catalyzed oxidations

Biocatalytic oxidation reactions are in high demand. Among the applied enzymes, the heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs), which relies on hydrogen peroxide as cosubstrate and oxidant, has generated great interest. Almost two decades after their first description, databases provide thousands of putative UPO sequences, but only a few enzymes have been characterized. To address this gap, efficient screening methods for the identification of novel UPOs are necessary. Here, a new screening strategy is presented based on solid cultivation of wild-type fungi. The identification of a promising candidate strain highlights the applicability of this approach

A gram-scale limonene production process with engineered escherichia coli

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

Biotechnological production of cyclic dinucleotides - challenges and opportunities

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

Investigation of vitamin D2 and vitamin D3 hydroxylation by Kutzneria albida

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

Comparative life cycle assessment of chemical and biocatalytic 2’3’-cyclic GMP-AMP synthesis

Life cycle assessments (LCAs) can provide insights into the environmental impact of production processes. In this study, a comparative LCA was performed for the synthesis of 2’3’-cyclic GMP-AMP (2’3’-cGAMP) in an early development stage. The cyclic dinucleotide (CDN) is of interest for pharmaceutical applications such as cancer immunotherapy. CDNs can be synthesized either by enzymes or chemical catalysis. It is not known which of the routes is more sustainable as both routes have their advantages and disadvantages, such as a poor yield for the chemical synthesis and low titers for the biocatalytic synthesis. The synthesis routes were compared for the production of 200 g 2’3’-cGAMP based on laboratory data to assess the environmental impacts. The biocatalytic synthesis turned out to be superior to the chemical synthesis in all considered categories by at least one magnitude, for example, a global warming potential of 3055.6 kg CO2 equiv. for the enzymatic route and 56454.0 kg CO2 equiv. for the chemical synthesis, which is 18 times higher. This study demonstrates the value of assessment at an early development stage, when the choice between different routes is still possible

Cell‐free protein synthesis for the screening of novel azoreductases and their preferred electron donor

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications

From coiled flow inverter to stirred tank reactor – bioprocess development and ontology design

Miniaturized bioreactors, such as the coiled flow inverter (CFI), offer several benefits within process development such as lower time and cost factors. In this study, we demonstrate continuous flow experiments in a CFI and transferred them to experiments in a batch reactor by using the oxygen transfer coefficient kLa as a key parameter. In order to simplify the parameter transfer and at the same time develop a basis for future data handling according to the FAIR data principles, an equipment and process ontology was developed for these examples

Catalytic promiscuity of cGAS: a facile enzymatic synthesis of 2′-3′-Linked cyclic dinucleotides

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

Application of cell-free protein synthesis for faster biocatalyst development

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