Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.

BACKGROUND: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. RESULTS: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. CONCLUSIONS: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.

Co-inoculation of antagonistic Bacillus velezensis FH-1 and Brevundimonas diminuta NYM3 promotes rice growth by regulating the structure and nitrification function of rhizosphere microbiome.

Microbial inoculation with plant growth-promoting microorganisms (PGPMs) is one of the most promising technologies to solve the current global challenges. Co-inoculants is more efficient and stable than mono-inoculants. However, the growth promoting mechanism of co-inoculants in complex soil system is still poorly understood. In this study, the effects on rice, soil and the microbiome of the mono-inoculant Bacillus velezensis FH-1 (F) and Brevundimonas diminuta NYM3 (N) and the co-inoculant FN obtained in previous works were compared. Correlation analysis and PLS-PM were used to explore the primary mechanism of different inoculants promoting rice growth. We hypothesized that inoculants promoted plant growth (i) by themselves, (ii) by improving soil nutrient availability or (iii) by regulating the rhizosphere microbiome in complex soil system. We also assumed that different inoculants had different ways of promoting plant growth. The results showed that FN significantly promoted rice growth and nitrogen absorption and slightly increased soil total nitrogen and microbial network complexity compared with F, N and the control (CK). B. velezensis FH-1 and B. diminuta NYM3 interfered with each other's colonization in FN. FN increased the complexity of the microbial network compared to F and N. The bacterial community of FN was quite different from CK and N, while the fungal community was not significantly different from other treatments. The species and functions enriched or inhibited by FN are part of F. The correlation analysis and PLS-PM results showed that inoculants (F/N/FN) promoted the growth of rice mainly by regulating the rhizosphere microbiome rather than by themselves or by improving soil nutrient availability. Co-inoculant FN promotes rice growth specifically by enhancing microbial nitrification function through enriching related species compared with F or N. This may provide theoretical guidance for the construction and application of co-inoculants in the future.

Sulfur vacancy and p-n junction synergistically boosting interfacial charge transfer and separation in ZnIn2S4/NiWO4 heterostructure for enhanced photocatalytic hydrogen evolution.

Constructing a p-n heterojunction with vacancy is advantageous for speeding up carrier separation and migration due to the synergy of the built-in electric field and electron capture of the vacancy. Herein, a sulfur vacancy riched-ZnIn2S4/NiWO4 p-n heterojunction (VZIS/NWO) photocatalyst was rationally designed and fabricated for photocatalytic hydrogen evolution. The composition and structure of VZIS/NWO were characterized. The existence of sulfur vacancy was confirmed through X-ray photoelectron spectroscopy, high-resolution transmission electron microscope, and electron paramagnetic resonance technology. The p-n heterojunction formed by ZnIn2S4 and NiWO4 was proved to provide a convenient channel to boost interfacial charge migration and separation. By reducing the band gap, the vacancy engineer can improve light absorption as well as serve as an electron trap to improve photo-induced electron-hole separation. Benefiting from the synergy of p-n heterojunction and vacancy, the optimal VZIS/NWO-5 catalyst exhibits dramatically enhanced H2 generation performance, which is about 10-fold that of the pristine ZnIn2S4. This work emphasizes the synergy between p-n heterojunction and sulfur vacancy for enhancing photocatalytic hydrogen evolution performance.

Multiple chemical valences induced interface regulation in perovskite nickelate/carbon nitride for boosting photocatalytic hydrogen evolution.

Recent developments in transition metal-based photocatalysts have heightened the need for superior solar utilization. Evidence suggests that properly adjusting the chemical valence of the transition metal elements could simultaneously achieve broad-spectrum absorption and efficient charge separation for the photocatalysts. However, the understanding and application of this strategy remain a significant challenge. Herein, a series of La0.9Ni0.8Co0.2O3-α/g-C3N4 (LNCO/CN) composites were synthesized employing a mild reduction procedure in the H2/Ar atmosphere. Experimental studies reveal that the composites regulated by interfacial coordination unsaturation Ni2+ and metal Ni0 possess accelerated Z-scheme charge transfer through the interfacial bond between Ni2+ and N. Besides, the localized-surface-plasmon-resonance-induced "hot electrons" injection process of in situ grown Ni0 nanoparticle is confirmed, which can efficiently quench the photoinduced holes and create hole vacancies around the interface. Due to the synergistic effect between Ni2+ and Ni0, the lifetime of the photo-excited electrons is prolonged with inhibited recombination behavior. After modulation, optimal LNCO/CN Z-scheme hybrid exhibits 9-fold promotion of photocatalytic hydrogen evolution rate compared to pristine LNCO/CN. This study gives valuable insight into the purposeful utilization of the chemical valence modulating strategy, which alters the chemical valence of transition metal elements to enhance the performance of perovskite-based photocatalysts dramatically.

Ultrafast interfacial charge evolution of the Type-II cadmium Sulfide/Molybdenum disulfide heterostructure for photocatalytic hydrogen production.

The interfacial charge dynamics was crucial for semiconductor heterostructure photocatalysis. Through the rational design of the heterostructure interface, heterojunction expressed variable recombination and migration dynamics for excited carriers. Herein, followed by a typical chemical bath strategy with the hexagonal cadmium sulfide (CdS) overlapped on the exfoliated molybdenum disulfide (MoS2) film, we developed a cadmium sulfide/molybdenum disulfide (CdS-MoS2) nano-heterojunction and investigated the interfacial charge dynamics for photocatalytic hydrogen evolution. Photoelectron spectroscopy detected an energetic offset between CdS and MoS2, revealing the formation of an interfacial electric field with efficient charges separation. Through transient absorption spectra, we demonstrated the type-II contact at the CdS-MoS2 interface. Driven by the electric field, the excited carriers separated and rapidly migrated to sub-band defects of CdS within the first 500 fs. The carriers-restricted defects provided catalytic active sites, endowing CdS-MoS2 a highly efficient photocatalytic capability. Consequentially, the CdS-MoS2 achieved an enhanced hydrogen evolution rate of 2.3 mmol·g-1·h-1 with significantly stronger photocurrent density. This work gave an insight to the channel of interfacial separation and migration for excited carriers, which could contribute to the interfacial engineering of advanced heterojunction photocatalysts.

Comprehensively dissecting the hub regulation of PkaC on high-productivity and pellet macromorphology in citric acid producing Aspergillus niger.

Aspergillus niger, an important industrial workhorse for citric acid production, is characterized by polar hyphal growth with complex pelleted, clumped or dispersed macromorphologies in submerged culture. Although organic acid titres are dramatically impacted by these growth types, studies that assess productivity and macromorphological changes are limited. Herein, we functionally analysed the role of the protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) signalling cascade during fermentation by disrupting and conditionally expressing the pkaC gene. pkaC played multiple roles during hyphal, colony and conidiophore growth. By overexpressing pkaC, we could concomitantly modify hyphal growth at the pellet surface and improve citric acid titres up to 1.87-fold. By quantitatively analysing hundreds of pellets during pilot fermentation experiments, we provide the first comprehensive correlation between A. niger pellet surface morphology and citric acid production. Finally, by intracellular metabolomics analysis and weighted gene coexpression network analysis (WGCNA) following titration of pkaC expression, we unveil the metabolomic and transcriptomic basis underpin hyperproductivity and pellet growth. Taken together, this study confirms pkaC as hub regulator linking submerged macromorphology and citric acid production and provides high-priority genetic leads for future strain engineering programmes.

Ferritin-Displayed GLP-1 with Improved Pharmacological Activities and Pharmacokinetics.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

Total Alkaloids from Bamboo Shoots and Bamboo Shoot Shells of Pleioblastus amarus (Keng) Keng f. and Their Anti-Inflammatory Activities.

The bamboo shoot of Pleioblastus amarus (Keng) Keng f. is a medicinal and edible plant product in China. In this study, the chemical composition of the total alkaloids from bamboo shoots and bamboo shoot shells of P. amarus (Keng) Keng f. (ABSP and ABSSP, respectively) were separated and investigated by UHPLC/QTOF-MS/MS. The results showed that a total of 32 alkaloids were extracted, with 15 common to both ABSP and ABSSP and 10 and 7 alkaloids distinct to ABSP and ABSSP, respectively. ABSP and ABSSP both decreased the lipopolysaccharide (LPS, 0.5 µg/mL)-induced nitric oxide (NO) production in RAW264.7 murine macrophages with half maximal inhibitory concentration (IC50) values of 78 and 55 µg/mL, respectively. We also found that ABSP and ABSSP (100 µg/mL) could decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both mRNA and protein levels in LPS-exposed RAW264.7 cells. Moreover, 100 µg/mL of ABSP and ABSSP also significantly inhibited LPS-induced mRNA expression of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α). Additionally, ABSP and ABSSP (100 µg/mL) decreased the phosphorylation of extracellular regulated protein kinase (ERK) in LPS-stimulated RAW264.7 cells. Collectively, the total alkaloids from the bamboo shoots and shells of P. amarus exhibit anti-inflammatory effects in LPS-activated RAW264.7 cells through the inhibition of ERK signaling. This result can provide support for the medicinal use and further study of P. amarus.

Facet Energy and Reactivity versus Cytotoxicity: The Surprising Behavior of CdS Nanorods.

Responsible development of nanotechnology calls for improved understanding of how nanomaterial surface energy and reactivity affect potential toxicity. Here, we challenge the paradigm that cytotoxicity increases with nanoparticle reactivity. Higher-surface-energy {001}-faceted CdS nanorods (CdS-H) were less toxic to Saccharomyces cerevisiae than lower-energy ({101}-faceted) nanorods (CdS-L) of similar morphology, aggregate size, and charge. CdS-H adsorbed to the yeast's cell wall to a greater extent than CdS-L, which decreased endocytosis and cytotoxicity. Higher uptake of CdS-L decreased cell viability and increased endoplasmatic reticulum stress despite lower release of toxic Cd(2+) ions. Higher toxicity of CdS-L was confirmed with five different unicellular microorganisms. Overall, higher-energy nanocrystals may exhibit greater propensity to adsorb to or react with biological protective barriers and/or background constituents, which passivates their reactivity and reduces their bioavailability and cytotoxicity.

Overexpression of profilin 3 affects cell elongation and F-actin organization in Arabidopsis thaliana.

KEY MESSAGE : Reduced levels of profilin 3 do not have a noticeable phenotypic effect; however, elevated profilin 3 levels result in decreased hypocotyl length due to a reduction in cell elongation and F-actin reorganization. The actin cytoskeleton is critical for a variety of cellular processes. The small actin monomer proteins, profilins (PRFs), are encoded by five highly conserved isoforms in Arabidopsis thaliana. PRF3, one of the vegetative isoforms, has 36 more N-terminal amino acid residues than the other four PRFs; however, the functions of PRF3 are mostly unknown. In this study, we demonstrated that PRF3 was strongly expressed in young seedlings, rosette leaves, and cauline leaves, but was weakly expressed in 14-day-old seedlings and flowers. Our data also showed that PRF3 could increase the critical concentration (Cc) of actin assembly in vitro. Overexpression of the full-length PRF3 cDNA resulted in a decrease in the lengths of roots and hypocotyls and delayed seed germination, but PRF3-ΔN36 transgenic plants and prf3 mutant plants showed normal growth when compared with wild-type plants. Microscopy observation revealed that cell elongation was inhibited in the hypocotyl and that F-actin was reorganized by destabilizing microfilaments. These results suggest that the dwarf phenotype of the PRF3 overexpression seedlings may be related to a reduction in cell length and F-actin rearrangement.