Biocatalysis is considered a green and environmentally friendly technology. Therefore, novel enzymes and enzymatic systems, together with cascades and protein engineering approaches, are in high demand. Here, three very different biocatalytic approaches have been studied. First, the richness of enzymes in the Red Sea brine pools has been assessed, and the discovery and characterization of a novel halophilic γ-carbonic anhydrase is described, together with the protein engineering approach, which boosted the initial catalytic activity of the γ- carbonic anhydrase. The understanding of polyextremophilicity principles from enzymes from the Red Sea brine pool, contributes to the bioengineering effort of turning mesophilic enzymes into more stable variants. Next, focus is given to the use of amine-transaminases in cascades for chiral amine synthesis. This resulted in the development of a self-sufficient sustainable cascade for chiral and non-chiral amine synthesis. This cascade was achieved by combining a lysine decarboxylase with an amine-transaminase to generate a cheap amino donor source for a more sustainable reaction economy. Finally, gas vesicle nanoparticles are functionalized by various engineering principles to create floating platforms for the immobilization of enzymes. The proof-of-concept was achieved by anchoring a phytase via anchoring peptides on the gas vesicle nanoparticles surface. These bioengineering approaches contributed to the effort of generating first principles for protein engineering.

Enzymes, as nature’s catalysts, show remarkable abilities that can revolutionize the chemical, biotechnological, bioremediation, agricultural and pharmaceutical industries. However, the narrow range of stability of the majority of described biocatalysts limits their use for many applications. To overcome these restrictions, extremozymes derived from microorganisms thriving under harsh conditions can be used. Extremophiles living in high salinity are especially interesting as they operate at low water activity, which is similar to conditions used in standard chemical applications. Because only about 0.1 % of all microorganisms can be cultured, the traditional way of culture-based enzyme function determination needs to be overcome. The rise of high-throughput next-generation-sequencing technologies allows for deep insight into nature’s variety. Single amplified genomes (SAGs) specifically allow for whole genome assemblies from small sample volumes with low cell yields, as are typical for extreme environments. Although these technologies have been available for years, the expected boost in biotechnology has held off. One of the main reasons is the lack of reliable functional annotation of the genomic data, which is caused by the low amount (0.15 %) of experimentally described genes. Here, we present a novel annotation algorithm, designed to annotate the enzymatic function of genomes from microorganisms with low homologies to described microorganisms. The algorithm was established on SAGs from the extreme environment of selected hypersaline Red Sea brine pools with 4.3 M salinity and temperatures up to 68°C. Additionally, a novel consensus pattern for the identification of γ-carbonic anhydrases was created and applied in the algorithm. To verify the annotation, selected genes were expressed in the hypersaline expression system Halobacterium salinarum. This expression system was established and optimized in a continuously stirred tank reactor, leading to substantially increased cell amounts and protein yields. The resulting gene expression products were assessed for function in vivo and/or in vitro. Our functional evaluation of the tested genes confirmed our annotation algorithm. Our developed strategy offers a general guide for using SAGs as a source of scientific and industrial investigations into “microbial dark matter” and may help to develop new catalysts, applicable for novel reactions in green chemistry.

Subsurface microbes are extremophiles adapted to thrive in deep, resource-limited environments, performing crucial roles in a myriad of biogeochemical processes. The extremozymes they produce might play a pivotal role in catalyzing these processes. Identifying and characterizing those enzymes could contribute to the advancements in industrially important biocatalytic reactions. Among various enzymes, sorbitol dehydrogenases are enzymes that catalyze the reversible conversion of sorbitol into fructose in the presence of NAD+. In this study, we focus on the exploration of a sorbitol dehydrogenase (SDHSIUC1) derived from the novel strictly anaerobic, thermophilic, subsurface bacterium, Caldiatribacterium inferamans SIUC1, which is one of the first cultured members from the candidate phylum Atribacteria OP9. As SDHSIUC1 originated from a subsurface microbe, we hypothesized that the enzyme has industrially beneficial characteristics such as higher thermostability and can be used for bioindustry applications such as synthesis of rare sugars and chiral alcohols. We successfully cloned, expressed, and purified the functional SDHSIUC1 enzyme aerobically using E. coli BL21(DE3) and did biochemical assays to characterize its properties. Additionally, in combination with the findings of biochemical characterization, we applied in silico approaches such as molecular modeling and molecular docking to describe the functional mechanism of the enzyme. Initial phylogenetic tree analysis using a pool of 24 amino acid sequences showed that the closest relative for SDHSIUC1 is a Candidatus Caldiatribacterium californiense, which is an uncultured member of the Atribacteria phylum. Size exclusion chromatography and Native-PAGE suggested that SDHSIUC1 is a hexamer with a size of 225 kDa. Kinetic characterization of the SDHSIUC1 showed that the enzyme has a higher affinity for sorbitol and fructose in the presence of NAD+ and NADH, respectively. Furthermore, SDHSIUC1 enzyme is promiscuous as it could utilize other polyols (i.e., glycerol, xylitol, inositol), diols (i.e., butanediol), aldehydes (i.e., glycolaldehyde), and ketoses (i.e., sorbose) in the presence of NAD+/NADH cofactors. We observed a significant increase in enzyme activity in the presence of Zn2+, where other metal ions such as Mn2+ and Mg2+ also resulted in rate improvements. The enzyme is an alkaline dehydrogenase that prefers a higher pH above 8. The effect of temperature on SDHSIUC1 activity showed that it’s a thermophilic enzyme with activity at 85 ℃. The thermal denaturation points of the enzyme at 85 ℃ was increased when the enzyme was preincubated at 85 ℃ in the presence of Zn2+. Notably, the enzyme preincubated 25 min at 85 ℃ in the presence of Zn2+ prefers fructose conversion and ceased the sorbitol conversion. We identified the presence of a structural Zn2+ binding site in SDHSIUC1 in addition to a catalytic Zn2+ binding site. We speculated that the structural Zn2+ involves thermal stability of the enzyme. Hence, we mutated the cysteine with serine of potential structural Zn2+ binding site (Cys96, Cys99, Cys102, and Cys110). Indeed, the Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed the mutated enzyme contains a lower amount of Zn2+ relative to the native enzyme. The data revealed that the mutated enzyme has low melting temperature (78 ℃) relative to the native enzyme (92 ℃), suggesting that structural Zn2+ is key to enhance the thermal stability of the SDHSIUC1. Surprisingly, we observed that the mutant enzyme completely lost its activity. The data suggests the role of structural Zn2+ binding site on both the structural and functional stability of SDHSIUC1. In consistence with the in-vitro data, the preliminary computational modeling data suggest that the losing structural Zn2+ unstable the enzyme and we are conducting in depth in-silico study to unveil the mechanism(s). We envisioned that the mechanisms behind the thermal stability of SDHSIUC1 could be used as basic model to enhance thermostable protein for the industrial application (e.g., design thermostable plastic hydrolyzing enzymes). To further demonstrate the potential applications of the SDHSIUC1, we genome-integrated it into the industrially important microorganism Pseudomonas putida KT2440. The resulting strain exhibited significantly increased growth in the presence of sorbitol compared to the wild-type P. putida KT2440, highlighting the potential of this enzyme for industrial applications such as enabling sorbitol catabolism or establishing xylose reductase pathway in P. putida KT2440 (i.e., leverage xylitol dehydrogenase activity of SDHSIUC1). In summary, this study has uncovered a novel thermostable sorbitol dehydrogenase from a subsurface microbe, which could have potential applications in the bioindustry where thermostable sorbitol dehydrogenases are required for the application in food and beverage industry, pharmaceutical industry, biofuel production etc. as it would be advantageous for the industrial processes.