Tailoring Pullulanase PulAR from Anoxybacillus sp. AR-29 for Enhanced Catalytic Performance by A Structure-Guided Consensus Approach

Pullulanase is a well-known debranching enzyme that can specially hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four sites (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase PulAR were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Four beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 5.0 and 6.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced via rational engineering by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

Streptomyces as Microbial Chassis for Heterologous Protein Expression

Heterologous production of recombinant proteins is gaining increasing interest in biotechnology with respect to productivity, scalability, and wide applicability. The members of genus Streptomyces have been proposed as remarkable hosts for heterologous production due to their versatile nature of expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes. However, there are several issues that limit their use, including low yield, difficulty in genetic manipulation, and their complex cellular features. In this review, we summarize rational engineering approaches to optimizing the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species in terms of genetic tool development and chassis construction. Further perspectives on the development of optimal Streptomyces chassis by the design-build-test-learn cycle in systems are suggested, which may increase the availability of secondary metabolites and recombinant proteins.

Structure-guided optimization of light-activated chimeric G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of GPCR function is the specific coupling of activated receptors to selected downstream signaling pathways. The ability to tune this coupling would permit the development of receptors with new capabilities. GPCRs and G-proteins have been recently resolved structurally at high resolution, but this information was in only very few cases harnessed for a rational engineering of these protein complexes. Here, we demonstrate the structure-guided optimization of coupling in chimeric light-activated GPCRs (OptoXRs). Our hypothesis was that the incorporation of structural GPCR-Gα contacts will lead to improved receptor activity. We first evaluated structure-based alignments as complements to existing sequence-based methods for generation of chimeric receptors. We then show in a prototypical light-activated β2AR that inclusion of α-helical residues forming structural contacts to Gα resulted in receptors with 7- to 20-fold increased function compared to other design strategies. In turn, elimination of GPCR-Gα contacts diminished function. Finally, the efficient receptor design served as a platform for the optimization of a further light-activated receptor and spectral tuning of the photoreceptor core domain. Our work exemplifies how increased OptoXR potency and new functionalities can be achieved through structure-based design towards targeted inputs into cells and cellular networks.

Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs

Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a red-shifted activation spectrum, cation selectivity, and large photocurrents while its slow closing kinetics impede some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests spectral and kinetic properties could be tuned with mutations and we identify ChRmine variants with two-fold increased and ten-fold decreased closing rates. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic manipulations.

Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

Background Developing a biologic medicine requires successful decision making during selection and optimization in addition to the pool of candidates at early research stages. Knowing structural information and binding patterns between drug target and discovery candidates greatly increases the probability of success. Methods With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated various tools in structural biology and the computation field and established a highly cost-effective platform which allows us to obtain fast and accurate structural information for nearly all our biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Results Here we report four case studies selected from 38 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered a challenge due to their small size. Conclusions Our utilization of this differential approach and multiple software packages allows us to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

SEARCH FOR RATIONAL EBGINEERING SOLUTIONS OF DIESEL LOCOMOTIVES FOR THE EASTERN RANGE

The purpose of the study is to search for rational engineering solutions for the main autonomous locomotive for the Eastern range of OAO Russian Railways. Research methods: methods of physical and field experiments, analytical methods for calculating the dynamics of the rolling stock. Research results and novelty: it is established that the use of a four-axle truck of a diesel locomotive TEM7 does not allow to create a locomotive that meets all the requirements of OAO Russian Railways, and the design of truck TEM7 is irrational for mainline locomotives; it is proved that the one-sided arrangement of brake blocks leads to deterioration in the locomotive braking properties. The bearing and axial towline, despite the use of an asynchronous engine, has an unsprung mass 1.5 times greater than that of the previously produced domestic analogue with a collector engine and almost the same mass of the wheel-motor unit; dynamic moments in the drive due to the lack of elastic elements during prolonged operation can reach 56% of the traction torque, which worsens the traction properties of the locomotive. Conclusion: it is advisable to carry out a design study of the drive variant with a support-frame asynchronous traction engine and an axial gearbox, as well as to conduct a technical and economic analysis and design study of a cheaper version of a diesel locomotive with sections on three two-axle bogies, maximally unified with electric locomotives, with a booster tanker module and AC-DC transmission with axial regulation of eight-pole collector motors with support-frame suspension.