Bioprocess development to produce a hyperthermostable S-methyl-5'-thioadenosine phosphorylase in Escherichia coli.

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5'-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1-90.1 g L-1 were observed together with an overproduction of ApMTAP in a 1.9%-3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg-1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.

Optimization of Inclusion Body Formation and Purification in Multi-well Plates.

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

A Comprehensive IT Infrastructure for an Enzymatic Product Development in a Digitalized Biotechnological Laboratory.

Typical product development in biotechnological laboratories is a distributed and versatile process. Today's biotechnological laboratory devices are usually equipped with multiple sensors and a variety of interfaces. The existing software for biotechnological research and development is often specialized on specific tasks and thus generates task-specific information. Scientific personnel is confronted with an abundance of information from a variety of sources. Hence a comprehensive software backbone that structures the developmental process and maintains data from various sources is missing. Thus, it is not possible to maintain data access, documentation, reporting, availability, and proper data exchange. This chapter envisions a comprehensive digital infrastructure handling the data throughout an enzymatic product development process in a laboratory. The platform integrates a variety of software products, databases, and devices to make all product development life cycle (PDLC) data available and accessible to the scientific staff.

Corrigendum: Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

[This corrects the article DOI: 10.3389/fbioe.2020.00854.].

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

Nucleoside-5'-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.