Precise genome editing of the model biocatalyst
Rhodococcus qingshengii
IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of
dsz
gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, CβS and MetB.
Rhodococcus
is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal.
Biodesulfurization (BDS) is considered a complementary technology to the traditional hydrodesulfurization treatment for the removal of recalcitrant sulfur compounds from petroleum products. BDS was investigated in a bubble column bioreactor using two-phase media. The effects of various process parameters, such as biocatalyst age and concentration, organic fraction percentage (OFP), and type of sulfur compound—namely, dibenzothiophene (DBT), 4-methyldibenzothiophene (4-MDBT), 4,6-dimethyldibenzothiophene (4,6-DMDBT), and 4,6-diethyldibenzothiophene (4,6-DEDBT)—were evaluated, using resting cells of Rhodococcus erythropolis IGTS8. Cells derived from the beginning of the exponential growth phase of the bacterium exhibited the highest biodesulfurization efficiency and rate. The biocatalyst performed better in an OFP of 50% v/v. The extent of DBT desulfurization was dependent on cell concentration, with the desulfurization rate reaching its maximum at intermediate cell concentrations. A new semi-empirical model for the biphasic BDS was developed, based on the overall Michaelis-Menten kinetics and taking into consideration the deactivation of the biocatalyst over time, as well as the underlying mass transfer phenomena. The model fitted experimental data on DBT consumption and 2-hydroxibyphenyl (2-HBP) accumulation in the organic phase for various initial DBT concentrations and different organosulfur compounds. For constant OFP and biocatalyst concentration, the most important parameter that affects BDS efficiency seems to be biocatalyst deactivation, while the phenomenon is controlled by the affinities of biodesulfurizing enzymes for the different organosulfur compounds. Thus, desulfurization efficiency decreased with increasing initial DBT concentration, and in inverse proportion to increases in the carbon number of alkyl substituent groups.
Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L−1, 58.6 μmole·min−1·(mg protein)−1, and 49 s−1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol−1·K−1, ΔH* = 37.0 kJ·mol−1 and ΔG* = 57.6 kJ·mol−1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*D, ΔG*D, ΔS*D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol−1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol−1·K−1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol−1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.
Synthetic biology applications rely on a well-characterized set of microbial strains, with an established toolbox of molecular biology methods for their genetic manipulation. Since there are no thermophiles with such attributes, most biotechnology and synthetic biology studies use organisms that grow in the mesophilic temperature range. As a result, thermophiles, a heterogenous group of microbes that thrive at high (>50 °C) temperatures, are largely overlooked, with respect to their biotechnological potential, even though they share several favorable traits. Thermophilic bacteria tend to grow at higher rates compared to their mesophilic counterparts, while their growth has lower cooling requirements and is less prone to contamination. Over the last few years, there has been renewed interest in developing tools and methods for thermophile bioengineering. In this perspective, we explain why it is a good idea to invest time and effort into developing a thermophilic synthetic biology direction, which is the state of the art, and why we think that the implementation of a thermophilic synthetic biology platform—a thermochassis—will take synthetic biology to the extremes.
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