Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

Genome and transcriptome sequencing of Trichoderma harzianum T4, an important biocontrol fungus of Rhizoctonia solani, reveals genes related to mycoparasitism.

Trichoderma harzianum is a well-known biological control strain and a mycoparasite of Rhizoctonia solani. To explore the mechanisms of mycoparasitism, the genome and transcriptome of T. harzianum T4 were both assembled and analyzed in this study. The genome of T. harzianum T4 was assembled into 106 scaffolds, sized 41.25 Mb, and annotated with a total of 8118 predicted genes. We analyzed the transcriptome of T. harzianum T4 against R. solani in a dual culture in three culture periods: before contact (BC), during contact (C), and after contact (AC). Transcriptome sequencing identified 1092, 1222, and 2046 differentially expressed genes (DEGs), respectively. These DEGs, which are involved in pathogen recognition and signal transduction, hydrolase, transporters, antibiosis, and defense-related functional genes, are significantly upregulated in the mycoparasitism process. The results of genome and transcriptome analysis indicated that the mycoparasitism process of T. harzianum T4 was very complex. T. harzianum successfully recognizes and invades host cells and kills plant pathogens by regulating various DEGs at different culture periods. The relative expression levels of the 26 upregulated DEGs were confirmed by RT-qPCR to validate the reliability of the transcriptome data. The results provide insight into the molecular mechanisms underlying T. harzianum T4's mycoparasitic processes, and they provide a potential molecular target for the biological control mechanism of T. harzianum T4.

The participation of vacuoles and the regulation of various metabolic pathways under acid stress promote the differentiation of chlamydospore in Trichoderma harzianum T4.

AIMS: Chlamydospores are a special, differentiated type with high environmental resistance. Consequently, the chlamydospores of Trichoderma harzianum T4 can used to industrialize the latter. This study aimed to investigate the key factors affecting the sporulation type of T. harzianum T4 and the mechanisms underlying this effect. METHODS AND RESULTS: In the liquid fermentation of T. harzianum T4, ammonium sulfate (AS) inhibited conidia formation and chlamydospore production. Fermentation tests revealed that acid stress induced sporulation type alteration. Transcriptomic analysis was used to evaluate the adaptation strategy and mechanism underlying spore type alteration under acid stress. The fermentation experiments involving the addition of amino acids revealed that branched-chain amino acids benefited conidia production, whereas ß-alanine benefited chlamydospore production. Confocal microscope fluorescence imaging and chloroquine intervention demonstrated that vacuole function was closely related to chlamydospore production. CONCLUSION: The sporulation type of T. harzianum T4 can be controlled by adjusting the fermentation pH. T. harzianum T4 cells employ various self-protection measures against strong acid stress, including regulating their metabolism to produce a large number of chlamydospores for survival.

Identification of Mycoparasitism-Related Genes against the Phytopathogen Botrytis cinerea via Transcriptome Analysis of Trichoderma harzianum T4.

Trichoderma harzianum is a well-known biological control agent (BCA) that is effective against a variety of plant pathogens. In previous studies, we found that T. harzianum T4 could effectively control the gray mold in tomatoes caused by Botrytis cinerea. However, the research on its biocontrol mechanism is not comprehensive, particularly regarding the mechanism of mycoparasitism. In this study, in order to further investigate the mycoparasitism mechanism of T. harzianum T4, transcriptomic sequencing and real-time fluorescence quantitative PCR (RT-qPCR) were used to identify the differentially expressed genes (DEGs) of T. harzianum T4 at 12, 24, 48 and 72 h of growth in the cell wall of B. cinerea (BCCW) or a sucrose medium. A total of 2871 DEGs and 2148 novel genes were detected using transcriptome sequencing. Through GO and KEGG enrichment analysis, we identified genes associated with mycoparasitism at specific time periods, such as encoding kinases, signal transduction proteins, carbohydrate active enzymes, hydrolytic enzymes, transporters, antioxidant enzymes, secondary metabolite synthesis, resistance proteins, detoxification genes and genes associated with extended hyphal longevity. To validate the transcriptome data, RT-qCPR was performed on the transcriptome samples. The RT-qPCR results show that the expression trend of the genes was consistent with the RNA-Seq data. In order to validate the screened genes associated with mycoparasitism, we performed a dual-culture antagonism test on T. harzianum and B. cinerea. The results of the dual-culture RT-qPCR showed that 15 of the 24 genes were upregulated during and after contact between T. harzianum T4 and B. cinerea (the same as BCCW), which further confirmed that these genes were involved in the mycoparasitism of T. harzianum T4. In conclusion, the transcriptome data provided in this study will not only improve the annotation information of gene models in T. harzianum T4 genome, but also provide important transcriptome information regarding the process of mycoparasitism at specific time periods, which can help us to further understand the mechanism of mycoparasitism, thus providing a potential molecular target for T. harzianum T4 as a biological control agent.

Exogenous Regulators Enhance the Yield and Stress Resistance of Chlamydospores of the Biocontrol Agent Trichoderma harzianum T4.

Trichoderma strains have been successfully used in plant disease control. However, the poor stress resistance of mycelia and conidia makes processing and storage difficult. Furthermore, they cannot produce chlamydospores in large quantities during fermentation, which limits the industrialization process of chlamydospore preparation. It is important to explore an efficient liquid fermentation strategy for ensuring chlamydospore production in Trichoderma harzianum. We found that the addition of mannitol, glycine betaine, and N-acetylglucosamine (N-A-G) during liquid fermentation effectively increases the yield of chlamydospores. Furthermore, we provided evidence that chlamydospores have stronger tolerance to high temperature, ultraviolet, and hypertonic stress after the addition of mannitol and trehalose. Lipids are an important component of microbial cells and impact the stress resistance of microorganisms. We studied the internal relationship between lipid metabolism and the stress resistance of chlamydospores by detecting changes in the lipid content and gene expression. Our results showed that mannitol and trehalose cause lipid accumulation in chlamydospores and increase the unsaturated fatty acid content. In conclusion, we verified that these exogenous regulators increase the production of chlamydospores and enhance their stress resistance by regulating lipid metabolism. In addition, we believe that lipid metabolism is an important part of the chlamydospore production process and impacts the stress resistance of chlamydospores. Our findings provide clues for studying the differentiation pathway of chlamydospores in filamentous fungi and a basis for the industrial production of chlamydospores.

Changes in Microbial Diversity, Soil Function, and Plant Biomass of Cotton Rhizosphere Soil Under the Influence of Chlorpyrifos.

Chlorpyrifos (CPF), a common organophosphorus pesticide, is extensively used in agricultural practices. However, we lack sound evidence for the linkage between soil microbial diversity, soil function, and plant biomass under the influence of CPF, which prevents us from assessing the actual impact of CPF on agricultural production. In this study, we used high-throughput sequencing to test the effects of CPF on soil microbial diversity, soil function, and cotton biomass in indoor pot experiments. The use of CPF leads to a significant reduction in cotton biomass until the concentration of CPF used reaches 15 mg kg-1, and the cotton biomass is no longer significantly reduced. Compared with the original soil, the alpha-diversity of bacteria, which was significantly linearly related to cotton biomass, was significantly decreased when the soil was treated with 15 mg kg-1 CPF. Affected by CPF, the overall soil microbial composition has changed significantly. Acidobacteria, Nitrospirae, Planctomycetes, and Actinobacteria were significantly regulated after CPF treatment. Correspondingly, key soil functions, including nitrogen metabolism and iron (III) transporter, have been significantly down-regulated. The reduction of nitrogen and Fe3+ should deprive the cotton of essential nutrients during the short crop cycle and thus affect cotton biomass. Our study provides experimental evidence that CPF affects cotton rhizosphere soil microbial diversity, the relative content of key bacterial genera, and soil function, which shows that it has an important impact on plant biomass, and provides a reference for studying the actual impact of CPF on the environment and agricultural production.

[Enzymatic properties of α-L-rhamnosidase and the factors affecting its activity: a review].

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.