Deciphering the stereo-specific catalytic mechanisms of cis-epoxysuccinate hydrolases producing L(+)-tartaric acid.

Microbial epoxide hydrolases, cis-epoxysuccinate hydrolases (CESHs), have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.

Composition of Lignocellulose Hydrolysate in Different Biorefinery Strategies: Nutrients and Inhibitors.

The hydrolysis and biotransformation of lignocellulose, i.e., biorefinery, can provide human beings with biofuels, bio-based chemicals, and materials, and is an important technology to solve the fossil energy crisis and promote global sustainable development. Biorefinery involves steps such as pretreatment, saccharification, and fermentation, and researchers have developed a variety of biorefinery strategies to optimize the process and reduce process costs in recent years. Lignocellulosic hydrolysates are platforms that connect the saccharification process and downstream fermentation. The hydrolysate composition is closely related to biomass raw materials, the pretreatment process, and the choice of biorefining strategies, and provides not only nutrients but also possible inhibitors for downstream fermentation. In this review, we summarized the effects of each stage of lignocellulosic biorefinery on nutrients and possible inhibitors, analyzed the huge differences in nutrient retention and inhibitor generation among various biorefinery strategies, and emphasized that all steps in lignocellulose biorefinery need to be considered comprehensively to achieve maximum nutrient retention and optimal control of inhibitors at low cost, to provide a reference for the development of biomass energy and chemicals.

Core-Shell Filament with Excellent Wound Healing Property Made of Cellulose Nanofibrils and Guar Gum via Interfacial Polyelectrolyte Complexation Spinning.

Natural polymer-based sutures have attractive cytocompatibility and degradability in surgical operations. Herein, anionic cellulose nanofibrils (ACNF) and cationic guar gum (CGG) are employed to produce nontoxic CGG/ACNF composite filament with a unique core-shell structure via interfacial polyelectrolyte complexation (IPC) spinning. The comprehensive characterization and application performance of the resultant CGG/ACNF filament as a surgical suture are thoroughly investigated in comparison with silk and PGLA (90% glycolide and 10% l-lactide) sutures in vitro and in vivo, respectively. Results show that the CGG/ACNF filament with the typical core-shell structure and nervation pattern surface exhibits a high orientation index (0.74) and good mechanical properties. The tensile strength and knotting strength of CGG/ACNF suture prepared by twisting CGG/ACNF filaments increase by 69.5%, and CGG/ACNF suture has a similar friction coefficient to silk and PGLA sutures. Moreover, CGG/ACNF suture with antibiosis and cytocompatibility exhibits better growth promotion of cells than silk suture, similar to PGLA suture in vitro. In addition, the stitching experiment of mice with the CGG/ACNF suture further confirms better healing properties and less inflammation in vivo than silk and PGLA sutures do. Hence, the CGG/ACNF suture with a simple preparation method and excellent application properties is promising in surgical operations.

Integrated lactic acid production from lignocellulosic agricultural wastes under thermal conditions.

The production of lactic acid (LA) from agricultural wastes attracts great attention because of the sustainability and abundance of lignocellulosic feedstocks, as well as the increasing demand for biodegradable polylactic acid. In this study, we isolated a thermophilic strain Geobacillus stearothermophilus 2H-3 for use in robust production of L-(+)LA under the optimal conditions of 60 °C, pH 6.5, which were consistent with the whole-cell-based consolidated bio-saccharification (CBS) process. Sugar-rich CBS hydrolysates derived from various agricultural wastes, including corn stover, corncob residue, and wheat straw, were used as the carbon sources for 2H-3 fermentation by directly inoculating 2H-3 cells into the CBS system, without intermediate sterilization, nutrient supplementation, or adjustment of fermentation conditions. Thus, we successfully combined two whole-cell-based steps into a one-pot successive fermentation process to efficiently produce LA with high optical purity (99.5%), titer (51.36 g/L), and yield (0.74 g/gbiomass). This study provides a promising strategy for LA production from lignocellulose through CBS and 2H-3 fermentation integration.

Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges.

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure or perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, various artificial small molecules have been successfully used in enzyme engineering. The types of enzymatic reactions and metabolic pathways in cells can be expanded by the incorporation of these artificial small molecules either as cofactors or as building blocks of proteins and nucleic acids, which greatly promotes the development and application of biotechnology. In this review, we summarized research on artificial small molecules including biological metal cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), and non-natural amino acids (nnAAs), focusing on their design, synthesis, and applications as well as the current challenges in synthetic biology.

Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a ß-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter -35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

Research advances on arachidonic acid production by fermentation and genetic modification of Mortierella alpina.

Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.

Structural Basis of Specificity for Carboxyl-Terminated Acyl Donors in a Bacterial Acyltransferase.

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy in overcoming multi-drug-resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a ß-lactamase homologue BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A and also exhibits substrate promiscuity toward acyl acceptors with different scaffolds. The crystal structure of BmmI covalently linked to the succinyl group and systematic mutagenesis highlighted the role of oxyanion holelike geometry in the recognition of carboxyl-terminated acyl donors. The engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. The structure of BmmI with acyl acceptor MLN A revealed the importance of Arg292 in the recognition of macrolide substrates. Moreover, the mechanism of the BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying the substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.

Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system.

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p- and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Solution structure of a unicellular microalgae-derived translationally controlled tumor protein revealed both conserved features and structural diversity.

Translationally controlled tumor proteins (TCTPs) are eukaryote-conserved multifunctional proteins, but their primary functions are not well understood yet. Study on TCTP from unicellular species may provide insight into the primary function of the TCTP family. Bioinformatic analysis indicated that the TCTP from Nannochloropsis oceanica (NoTCTP), a model unicellular microalga for biodiesel and polyunsaturated fatty acid production, has low sequence homology to other structure-known TCTPs and two TCTP signature patterns are not detected in NoTCTP. Hence, we determined the solution structure of NoTCTP. The overall structure of NoTCTP, including a long flexible loop, a ß-barrel subdomain, and a helical subdomain, is generally similar to other TCTP structures. NoTCTP has a eukaryote-conserved surface for the binding of eukaryotic elongation factor 1B, confirming that TCTP is involved in protein synthesis, which is one of the primary functions of TCTP. Additionally, NoTCTP has distinct features different from other TCTPs. NoTCTP structure lacks a short α-helix which exists in all other known TCTP structures. The helical subdomain and some loops of NoTCTP also have distinct conformations among the TCTP family proteins. Therefore, our study on NoTCTP revealed not only conserved structural features but also the structural diversity of TCTP family proteins.

An Effective Strategy for Identification of Highly Unstable Bacillaenes.

Exploration of unstable compounds is a rarely explored area of natural product research. We describe the integration of genomic and metabolomic analyses with bioassay-guided compound mining to effectively explore unstable bacillaenes. New bacillaene structures (2, 4, and 5) were identified from compound mixtures using the DANS-SVI (differential analysis of 2D NMR spectrum-single spectrum with variable intensities) method, which were further verified by the isolation of the pure compounds under strictly controlled conditions. Compound 1 exhibited antibacterial activity against multi-drug-resistant bacterial strains, while glycosylation decreased the activity of the bacillaene scaffold.

Diagnostic Value of Arrival Time Parametric Imaging Using Contrast-Enhanced Ultrasonography in Superficial Enlarged Lymph Nodes.

OBJECTIVE: The aim of this study was to evaluate the value of arrival-time parametric imaging for differential diagnosis of superficial enlarged lymph nodes. METHODS: Patients with lymphadenopathy who received contrast-enhanced ultrasonography (CEUS) and biopsy were included in this study. Following CEUS, a prototype software of the arrival-time parametric imaging system was used to analyze the video footage. Arrival-time patterns during the arterial phase were evaluated. The quantitative parameters including arrival time of periphery, arrival time of center, and the travel time (△T) were calculated. RESULTS: A total of 145 lymph nodes were analyzed. Arrival-time parametric imaging showed that 80.3% of metastatic lymph nodes and 68.4% of lymphoid tuberculosis presented a centripetal perfusion pattern, 76.5% of lymphoma showed complete homogeneous enhancement, and 81.2% of reactive lymph nodes had centrifugal patterns. The arrival time of periphery (sec) of metastatic lymph nodes was substantially earlier than that of lymphoma (11.0 ± 3.1 versus 12.6 ± 3.6; P < .05). The arrival time of center (sec) of metastatic lymph nodes was obviously later than that of lymphoma and reactive lymph nodes (13.4 ± 3.3 versus 10.5 ± 2.9 and 10.6 ± 1.5; P < .05). The travel time (△T) (sec) in metastatic lymph nodes was substantially longer than in reactive lymph nodes and lymphoma (4.2 ± 2.1 versus 2.3 ± 1.6 and 2.9 ± 2.5; P < .05). At a △T cutoff value of 2.75 seconds (using the receiver operating characteristic curve), the sensitivity and specificity in differentiating metastatic lymph nodes from benign lymph nodes (lymphoid tuberculosis and reactive lymph nodes) were 78.9% and 64.7%, respectively. CONCLUSIONS: Enhanced patterns and parameters of arrival-time parametric imaging during CEUS could provide more information for the differential diagnosis of enlarged superficial lymph nodes.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

BACKGROUND: Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. RESULTS: The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L- 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. CONCLUSIONS: This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Marinobacter bohaiensis sp. nov., a moderate halophile isolated from benthic sediment of the Bohai Sea.

A Gram-stain-negative, motile, aerobic and rod-shaped bacterial strain, designated T17T, was isolated from benthic sediment sampled at Jiaozhou Bay, Bohai Sea, China, and its taxonomic position was investigated. The 16S rRNA gene sequence of strain T17T exhibited the highest similarity values to those of the type strain Marinobacter lacisalsi FP2.5 (96.2 %) and Marinobacter koreensis DD-M3T (96.2 %). Strain T17T grew optimally at 35 °C, pH 7.0-8.0 and in the presence of 6.0-10.0 % (w/v) NaCl. The predominant ubiquinone in strain T17T was identified as Q-9. The major fatty acids of strain T17T were C12 : 0, C16 : 0 and C16 : 0 10-CH3. The major polar lipids of strain T17T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidglycerol, an unidentified aminolipid and an unidentified phospholipid. The DNA G+C content of strain T17T was 63.0 mol%. The draft genome sequence of strain T17T includes 4 755 891 bp in total (N50=2 856 325 bp) with a medium read coverage of 100.0x and 11 scaffolds. In silico DNA-DNA hybridization with the three type strains showed 20.3, 19.7 and 19.9 % relatedness to Marinobacter santoriniensis NKSG1T, Marinobacter segnicrescens SS11B1-4T and Marinobacter daqiaonensis CGMCC 1.9167T, respectively. On the basis of the phenotypic, phylogenetic, genomic and chemotaxonomic properties, strain T17T is considered to represent a novel species within the genus Marinobacter, for which the name Marinobacterbohaiensis sp. nov. is proposed. The type strain is T17T (=KCTC 52710T=MCCC 1K03282T).

Diagnostic Value of Contrast-Enhanced Ultrasonography and Time-Intensity Curve in Differential Diagnosis of Cervical Metastatic and Tuberculous Lymph Nodes.

OBJECTIVES: To evaluate the diagnostic value of contrast-enhanced ultrasonography (CEUS) in the differential diagnosis of tuberculous and metastatic lymph nodes. METHODS: Nineteen cervical tuberculous lymph nodes and 43 cervical metastatic lymph nodes were investigated. The CEUS perfusion patterns and parameters of time-intensity curve (TIC) were analyzed. Diagnostic accuracy and consistency of two physicians were compared before and after CEUS and TIC analysis. RESULTS: Conventional ultrasonography showed no significant differences between tuberculous and metastatic lymph nodes. Concentric enhancement at the arterial phase of CEUS occurred in 15 of 19 (78.9%) tuberculous lymph nodes and 42 of 43 (97.7%) metastatic lymph nodes (P < .05). For the TIC curve, a steep descending curve with an apparent notch was commonly found in tuberculous lymph nodes (13 of 16). Although a shallow descending curve was common (40 of 43) in metastatic lymph nodes, most did not have a notch on the curve (39 of 43) (P < .01). The k-value and the peak intensity (PI) value of tuberculous lymph nodes were significantly higher and the △PI value was significantly lower than that of metastatic lymph nodes (P < .05, respectively). Kappa values for the diagnosis consistency of the two physicians before and after CEUS and TIC analysis were 0.582 and 0.761, respectively. Diagnostic accuracy before and after CEUS and TIC analysis was 47.4% (28 of 59) and 96.6% (57 of 59), respectively (P < .001). CONCLUSIONS: Contrast-enhanced ultrasonography with TIC analysis is helpful in differentiating tuberculous from metastatic lymph nodes.

Structural Insight into the Stabilizing Effect of O-Glycosylation.

Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far, the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide are valuable steps toward being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule's physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.

Expression of Vitreoscilla hemoglobin enhances production of arachidonic acid and lipids in Mortierella alpina.

BACKGROUND: Arachidonic acid (ARA, C20:4, n-6), which belongs to the omega-6 series of polyunsaturated fatty acids and has a variety of biological activities, is commercially produced in Mortierella alpina. Dissolved oxygen or oxygen utilization efficiency is a critical factor for Mortierella alpina growth and arachidonic acid production in large-scale fermentation. Overexpression of the Vitreoscilla hemoglobin gene is thought to significantly increase the oxygen utilization efficiency of the cells. RESULTS: An optimized Vitreoscilla hemoglobin (VHb) gene was introduced into Mortierella alpina via Agrobacterium tumefaciens-mediated transformation. Compared with the parent strain, the VHb-expressing strain, termed VHb-20, grew faster under both limiting and non-limiting oxygen conditions and exhibited dramatic changes in cell morphology. Furthermore, VHb-20 produced 4- and 8-fold higher total lipid and ARA yields than those of the wild-type strain under a microaerobic environment. Furthermore, ARA production of VHb-20 was also 1.6-fold higher than that of the wild type under normal conditions. The results demonstrated that DO utilization was significantly increased by expressing the VHb gene in Mortierella alpina. CONCLUSIONS: The expression of VHb enhances ARA and lipid production under both lower and normal dissolved oxygen conditions. This study provides a novel strategy and an engineered strain for the cost-efficient production of ARA.

Amylibacter cionae sp. nov., isolated from the sea squirt Ciona savignyi.

A Gram-stain-negative, non-motile, aerobic and rod-shaped bacterial strain, designated H-12T, was isolated from a sea squirt (Ciona savignyi) collected from Tsingtao Port, Jiaozhou Bay, China, and its taxonomic position was investigated. Strain H-12T grew optimally at 25-30 °C, at pH 7.0-8.0 and in the presence of 3.0-4.0 % (w/v) NaCl. The 16S rRNA gene sequence of strain H-12T exhibited the highest similarity to that of the type strain of Amylibacter marinus (95.3 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain H-12T clustered with the type strain of A. marinus. The predominant ubiquinone in strain H-12T was identified as Q-10. The major fatty acids of strain H-12T were C18 : 1ω7c and C18 : 1ω7c 11-methyl. The major polar lipids detected in strain H-12T were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, one unidentified aminolipid, two unidentified phospholipids and five unidentified lipids. The DNA G+C content of strain H-12T was 52.7 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic properties, strain H-12T is considered to represent a novel species within the genus Amylibacter, for which the name Amylibacter cionae sp. nov. is proposed. The type strain is H-12T (=KCTC 52581T=CGMCC 1.15880T).

Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes.

Revisiting the NMR solution structure of the Cel48S type-I dockerin module from Clostridium thermocellum reveals a cohesin-primed conformation.

Dockerin modules of the cellulosomal enzyme subunits play an important role in the assembly of the cellulosome by binding tenaciously to cohesin modules of the scaffoldin subunit. A previously reported NMR-derived solution structure of the type-I dockerin module from Cel48S of Clostridium thermocellum, which utilized two-dimensional homonuclear (1)H-(1)H NOESY and three-dimensional (15)N-edited NOESY distance restraints, displayed substantial conformational differences from subsequent structures of dockerin modules in complex with their cognate cohesin modules, raising the question whether the source of the observed differences resulted from cohesin-induced structural rearrangements. Here, we determined the solution structure of the Cel48S type-I dockerin based on (15)N- and (13)C-edited NOESY-derived distance restraints. The structure adopted a fold similar to X-ray crystal structures of dockerin modules in complex with their cohesin partners. A unique cis-peptide bond between Leu-65 and Pro-66 in the Cel48S type-I dockerin module was also identified in the present structure. Our structural analysis of the Cel48S type-I dockerin module indicates that it does not undergo appreciable cohesin-induced structural alterations but rather assumes an inherent calcium-dependent cohesin-primed conformation.