TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.

Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3-5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases6-8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases.

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids.

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant.

Usage of GD-95 and GD-66 lipases as fusion partners leading to improved chimeric enzyme LipGD95-GD66.

Lipases are used as biocatalysts in industrial processes mainly because of their stability at broad temperature and pH range, resistance to organic solvents and wide spectrum of substrates. The usage of several lipolytic domains, each with different activity and resistance profiles, enables both the flexibility and efficiency of industrial processes. In this study, GD-95 and GD-66 lipases produced by Geobacillus sp. 95 and Geobacillus sp. 66, respectively, were used as fusion partners to create a new fused lipolytic enzyme LipGD95-GD66. Chimeric LipGD95-GD66 lipase displayed tenfold increase in activity (200 U/mg) compared to parental GD-66 lipase, improved Vmax (10 µmol/min mg-1) and catalytic efficiency (2 ∗ 105 min-1 mM-1) for p-NP palmitate as a substrate and increased activity at 70-75 °C compared to both parental lipases. All three lipases also retained >50% of their lipolytic activity after incubation with methanol, n-hexane, ethanol and DMF for longer than three weeks, highlighting a great prospect for application in industrial processes. Moreover, transesterification results revealed the capability of parental GD-95 lipase to be the most promising biocatalyst for production of methyl and ethyl esters through eco-friendly transesterification using argan oil and ethanol/methanol as acceptors of acyl group.