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.

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.

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.The chemical synthesis of 2 3 -cGAMP contains eight reaction steps and suffers from low yields [8,9], whereas the enzymatic reaction reaches nearly full conversion within a few hours [6]. In recent studies, human cGAS was investigated predominantly with regards to the identification of key residues [10,11] and the kinetic mechanism [12]. Although several homologous enzymes are known, only murine cGAS [6,7], porcine cGAS [10,13], and chicken cGAS [14] received more attention. For the murine and porcine homologue, crystal structures are also available and, in parts, crucial amino acids with relevance for the catalytic function are known. Alignments of amino acid sequences of identified cGAS homologues revealed a low-sequence homology in regions without determined function [10,15]. This high level of variation and the markedly reduced activity of human cGAS in comparison to murine cGAS [11] demonstrate the possibility that other cGAS homologues might be available with enhanced properties or different characteristics and functions. For characterization, cGAS enzymes were synthesized with eukaryotic cell lines or expressed recombinantly in Escherichia coli. Eukaryotic genes tend to be difficult to express in bacterial systems, though fusion-tags, such as maltose-binding protein (MBP, 42.5 kDa) [16], glutathione S-transferase (GST, 26 kDa) [2], and small ubiquitin-like modifier (SUMO, 12 kDa) [1], were used for better solubility and functional expression of cGAS. Challenges remain to find suitable expression systems for eukaryotic protein production with good yield and purity, and under conditions conducive to functional protein studies. To realize higher throughput in heterologous protein synthesis, other methods than traditional recombinant expression can be considered.

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.

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.

The selective hydroxylation of non-activated C-H bonds is still a challenging reaction in chemistry. Non-heme Fe2+/α-ketoglutarate-dependent dioxygenases are remarkable biocatalysts for the activation of C-H-bonds, catalyzing mainly hydroxylations. The discovery of new Fe2+/α-ketoglutarate-dependent dioxygenases with suitable reactivity for biotechnological applications is therefore highly relevant to expand the limited range of enzymes described so far. In this study, we performed a protein BLAST to identify homologous enzymes to already described lysine dioxygenases (KDOs). Six novel and yet uncharacterized proteins were selected and synthesized by cell-free protein synthesis (CFPS). The subsequent in vitro screening of the selected homologs revealed activity towards the hydroxylation of l-lysine (Lys) into hydroxy-l-lysine (Hyl), which is a versatile chiral building block. With respect to biotechnological application, Escherichia coli whole-cell biocatalysts were developed and characterized in small-scale biotransformations. As the whole-cell biocatalyst expressing the gene coding for the KDO from Photorhabdus luminescens showed the highest specific activity of 8.6 ± 0.6 U gCDW−1, it was selected for the preparative synthesis of Hyl. Multi-gram scale product concentrations were achieved providing a good starting point for further bioprocess development for Hyl production. A systematic approach was established to screen and identify novel Fe2+/α-ketoglutarate-dependent dioxygenases, covering the entire pathway from gene to product, which contributes to accelerating the development of bioprocesses for the production of value-added chemicals.

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.

Cell‐free protein synthesis (CFPS) systems are an attractive method to complement the usual cell‐based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well‐established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.