Function-based searches for selected phosphotransferases and establishing in vitro transcription platform for cell-free metagenomics

Abstract

The shift in industrial production of specialty chemicals, pharmaceuticals, and metabolites towards biocatalysis has been the center of interest for several decades. The progress, however, is largely challenged due to the limited enzyme diversity to fulfil synthetic requirements. This has channeled a significant effort towards screening enzymes with unique features especially from extreme environments. While this has a huge potential to expand the enzyme toolkits, the existing functional screening methods are severely limited in tapping nature’s unlimited potential. The aim of this study was to search for selected phosphotranferases, including rare and difficult-to-express enzymes, for use in biocatalytic phosphorylation of target metabolites. It thereby aimed at establishing in vitro transcription system to lay the foundation for cell-free functional metagenomic screening platform. Functional screening of enzymes for phosphorylation of glycerol and some of its derivatives is presented here. While a previous report on glycerol-2-phosphotransferse activity was challenged, functional search of ß-glycerophosphate-involving activities were screened from environmental samples after enrichment. The use of a highly enantio-selective kinase integrated with energy regeneration system was also established for sustainable biocatalytic production of optically pure D-glycerate-2-phosphate and product analysis thereafter. Additionally, multiple expression attempts of acid-stable glycerol kinases from the most extreme acidophilic microbes ever reported was performed for biocatalytic synthesis of the very labile glyceraldehyde-3-phosphate. Alongside, engineering process parameters led to the use of an existing kinase for efficient synthesis of L-glyceraldehyde-3-phosphate which suggests to strategically consider adapting available enzymes for desired applications. In addition to the successes achieved here, the search for phosphotransferases faced a serious challenge of protein expression. Not surprisingly, any function-based screening approach including metagenomics suffers a similar impediment where difference in codon usage often leads to biased transcription ultimately resulting poor protein expression. To this end, an in vitro transcription system based on a recombinant RNA polymerase (RNAP) from Geobacillus sp. GHH01 was established. The RNAP can easily be reconstituted using a new ‘thermo-reconstitution’ method which requires only a brief thermal treatment saving time and resources compared to the classical method of reconstitution. One of the striking features of this RNAP is its ability to successfully initiate transcription from diverse recognition factors as evidenced by the amount of transcript generated when employing meta/genomic DNA templates from different environmental samples and organisms of varying phylogenetic origins. While phage-derived RNAPs are limited due to their absolute stringency towards own recognition factors, the Geobacillus RNAP has a significant comparative advantage over the tested commercial Escherichia coli RNAP in terms of both the transcript generated and other important features. The Geobacillus RNAP has remarkable working and storage stabilities maintaining its maximum activity up to 55°C and can simply be stored at non-freezing temperatures. In this work, cell-free metagenomic screening and expression platform is suggested to overcome the multitude of challenges associated with host systems used for functional metagenome searches.