The protein methyltransferase TrSAM inhibits cellulase gene expression by interacting with the negative regulator ACE1 in Trichoderma reesei.

Protein methylation is a commonly posttranslational modification of transcriptional regulators to fine-tune protein function, however, whether this regulation strategy participates in the regulation of lignocellulase synthesis and secretion in Trichoderma reesei remains unexplored. Here, a putative protein methyltransferase (TrSAM) is screened from a T. reesei mutant with the ability to express heterologous ß-glucosidase efficiently even under glucose repression. The deletion of its encoding gene trsam causes a significant increase of cellulase activities in all tested T. reesei strains, including transformants of expressing heterologous genes using cbh1 promotor. Further investigation confirms that TrSAM interacts with the cellulase negative regulator ACE1 via its amino acid residue Arg383, which causes a decrease in the ACE1-DNA binding affinity. The enzyme activity of a T. reesei strain harboring ACE1R383Q increases by 85.8%, whereas that of the strains with trsam or ace1 deletion increases by more than 100%. By contrast, the strain with ACE1R383K shows no difference to the parent strain. Taken together, our results demonstrate that TrSAM plays an important role in regulating the expression of cellulase and heterologous proteins initiated by cbh1 promotor through interacting with ACE1R383. Elimination and mutation of TrSAM and its downstream ACE1 alleviate the carbon catabolite repression (CCR) in expressing cellulase and heterologous protein in varying degrees. This provides a new solution for the exquisite modification of T. reesei chassis.

Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method.

Claviceps purpurea produces many pharmacologically important ergot alkaloids (EAS), which are widely used to treat migraine and hypertension and to aid childbirth. Although an EAS biosynthetic cluster of C. purpurea has been discovered more than 20 years ago, the complete biosynthetic pathway of EAS has not been fully characterized until now. The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C. purpurea. The conventional gene manipulation method for C. purpurea relies on homologous recombination (HR), although the efficiency of HR in C. purpurea is very low (∼1-5%). Consequently, the disruption of target genes is laborious and time-consuming. Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently, their gene-disruption efficiency is still very low. Here, we developed an efficient genome-editing system in C. purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes. As proof of principle, three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system, with editing efficiencies ranging from 50% to 100%. Inactivation of the three genes, which are closely related to uridine biosynthesis (ura5), hypha morphology (rac), and EAS production (easA), resulted in a uridine auxotrophic mutant, a mutant with a drastically different phenotype in axenic culture, and a mutant that did not produce EAS, respectively. Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C. purpurea, which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C. purpurea.

High-level sustainable production of the characteristic protopanaxatriol-type saponins from Panax species in engineered Saccharomyces cerevisiae.

The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C6-O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.

Alleviating product inhibition of Trichoderma reesei cellulase complex with a product-activated mushroom endoglucanase.

Product inhibition of cellulase is a challenging issue in industrial processes. Here, we introduced a product-activated mushroom cellulase, PaCel3A from Polyporus arcularius, into Trichoderma reesei. The filter paper activity, carboxymethyl cellulase activity, and saccharification efficiency (substrate: pretreated rice straw, PRS) of transformants increased significantly with this enzyme (by 18.4-26.8%, 13.8-22.8%, and 17.0%, respectively). A mutant of PaCel3A, PaCel3AM, obtained based on B-factor analysis, saturated mutagenesis, and residual activity assay, showed improved thermostability. The PRS saccharification efficiency using the cellulase complex from T. reesei transformants overexpressing pacel3am increased by 56.4%-63.0%. In addition, the T. reesei cellulase complex obtained by adding the purified recombinant PaCel3AM from T. reesei (rCel3aM-tr) to hydrolyze PRS resulted in increased reducing sugar yields at all sampling points, outperforming the cellulase complexes without rCel3aM-tr. These results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.

Expanded study on the risk of lymphovascular space invasion and lymph node metastasis of endocervical adenocarcinoma using Pattern Classification: a single-centre analysis of 213 cases.

A new three-tiered Pattern Classification system for usual-type endocervical adenocarcinomas (U-EACs) recommends using tumour invasive patterns rather than depth of invasion (DOI) and horizontal spread to categorise tumours. Tumours categorised by Pattern Classification are associated with lymph node (LN) metastasis and adverse outcomes. The aim of this study is to further explore the potential of Pattern Classification in surgical pathology practice. A total of 213 consecutive cases [201 U-EACs and 12 gastric-type adenocarcinomas (GACs)] diagnosed between 2006 and 2017 was retrospectively analysed. Clinicopathological data included age at diagnosis, DOI measurement, the status of lymphovascular space invasion (LVSI) and LN metastasis, and the number of LVSI foci, dissected and metastatic LNs. Immunostaining for CD34 and D2-40 was performed to identify LVSI in 14 challenging cases. Overall, mean age at diagnosis was 51 years (range 23-75). LVSI and LN metastasis occurred in 128 (60.1%, 128/213) and 42 (20.5%, 42/205) cases, respectively. Also, 28 (13.1%), 21 (9.9%), and 164 (77.0%) patients had pattern A, B, and C tumours, respectively. Patients with pattern C tumours had the oldest age at diagnosis (p=0.007), the highest incidence of LVSI and LN metastasis, and the highest DOI (p<0.001). Due to a highly heterogeneous growth pattern, pattern C U-EACs were stratified into four subgroups: C1, C2 and C3 corresponded to solid, extensive linear destructive, and band-like lymphocytic infiltrate growth patterns, respectively, and C4 included diffuse destructive, confluent, micropapillary and mixed growth pattern. C2 and C3 subgroup tumours had lower incidence of LVSI (20% and 40%, respectively) than the other two subgroups (p<0.001). None of the patients with C2 and C3 subgroup had LN metastasis and ≥3 LVSI foci. All GACs belonged to pattern C and had deeper stromal invasion (p=0.008), higher incidence of LN metastasis (p=0.001), and larger quantity of LVSI foci (p=0.008) and metastatic LNs (p=0.004) than those of pattern C U-ECAs. Number of LVSI foci were moderately positively correlated with LN metastasis status (p<0.001, γ=0.489) or number of metastatic LNs (p<0.001, γ=0.409). Our study further supports that Pattern Classification is a system easy to follow, which has a strong correlation to LVSI and an effective predictability for LN metastasis. Extensive linear destructive and band-like lymphocytic infiltrate growth patterns in pattern C U-EACs need to be recognised, as they behave less aggressively than that for the other growth pattern subgroups. Our study supports that Pattern Classification can be routinely applied to guide therapies for patients with U-EACs.

Competitive molecular-/ion-recognition responsive characteristics of poly(N-isopropylacrylamide-co-benzo-12-crown-4-acrylamide) copolymers with benzo-12-crown-4 as both guest and host units.

Novel dual molecular- and ion-recognition responsive poly(N-isopropylacrylamide-co-benzo-12-crown-4-acrylamide) (PNB12 C4 ) linear copolymers with benzo-12-crown-4 (B12C4) as both guest and host units are prepared. The copolymers exhibit highly selective sensitivities toward γ-cyclodextrin (γ-CD) and Na(+) . The presence of γ-CD induces the lower critical solution temperature (LCST) of PNB12 C4 copolymer to shift to a higher value due to the formation of 1:1 γ-CD/B12C4 host-guest inclusion complexes, while Na(+) causes a negative shift in LCST due to the formation of 2:1 "sandwich" B12C4/Na(+) host-guest complexes. Regardless of the complexation order, when γ-CD and Na(+) coexist with PNB12 C4 , competitive complexation actions of B12C4 as both guest and host units toward γ-CD and Na(+) finally form equilibrium 2:2:1 γ-CD/B12C4/Na(+) composite complexes, and the final LCST values of PNB12 C4 copolymer reach almost the same level. The results provide valuable guidance for designing and applying PNB12 C4 -based smart materials in various applications.

A metal-decorated nickel foam-inducing regulatable manganese dioxide nanosheet array architecture for high-performance supercapacitor applications.

Three dimensional manganese dioxide/Pt/nickel foam (shortened to MnPtNF) hybrid electrodes were prepared by double-pulse polarization and potentiostatic deposition technologies for supercapacitor applications. The decoration of Pt nanoparticles onto nickel foam varies the nucleation mechanism of the manganese dioxide species, inducing the formation of manganese dioxide nanosheets. Additionally, controlling the size of the Pt nanoparticles leads to modulated nanosheet architecture and electrochemical properties of the manganese dioxide electrode, as revealed by XRD, Raman spectra, SEM, TEM, cyclic voltammetry, galvanostatic charge-discharge and electrochemical impedance spectroscopy. The nanosheet architecture of the MnPtNF electrode favors the transportation of electrons and ions, which results in the enhanced electrochemical properties. Importantly, the optimized MnPtNF electrode obtains a maximum specific capacitance of 1222 F g(-1) at 5 A g(-1) (89% of the theoretical specific capacitance of MnO2) and 600 F g(-1) at 100 A g(-1). Moreover, the presence of Pt nanoparticles in the MnO2 electrode effectively improves its cycling stability, which is confirmed by the increase of the specific capacitance retention from 14.7% to 90% after 600 cycles.

Comparison of the expression of 5 heat shock proteins in benign and malignant salivary gland tumor tissues.

The objective of this study was to analyze the significance and potential value of heat shock proteins (HSPs) in salivary gland tumors. We found that expression of HSP60, HSP70, HSP86 and HSP84 were all upregulated in both salivary gland benign tumors and malignant tumors, and that the expression of HSP70, HSP86 and HSP84 was more greatly overexpressed in the malignant tumors (each P<0.01). For HSP27, expression was upregulated both in malignant and benign tumors, with less expression observed in malignant tumors (P<0.01). In malignant tumors, expression of HSP27 was negatively correlated with the age of the patients, size of the tumor tissue, occurrence of neural invasion and metastasis (each P<0.05). Additionally, in malignant tumors, HSP70 and HSP86 were both positively correlated with occurrence site, neural invasion and metastasis (each P<0.05), while HSP60 was only negatively correlated with the age of the patients (P<0.05). HSP86 was also positively correlated with malignant degree (P<0.01). In malignant tumors, the proliferation index (PI), which was marked by proliferating cell nuclear antigen (PCNA; PCNA-PI) was 49.95±14.569, which was significantly higher compared with that in benign tumors (P<0.001), which was in accordance with the upregulation of HSP70, HSP86 or HSP84; however, an adverse correlation was found between HSP27 expression and PCNA (each P<0.05). In conclusion, these results suggest that HSPs are involved in the occurrence and development of salivary gland tumors. HSP70, HSP86 and HSP84 retained the higher multiplication capability of the malignant tumor cells, however, HSP27 did not. Thus, the upregulation of HSP70, HSP86 and HSP84 and the downregulation of HSP27 may all be used as biomarkers of the occurrence and development of malignant salivary gland tumors. Moreover, the extremely high expression of HSP86 and HSP84 in benign tumors indicates the malignant transformation potential.