Lingyan Dai

To increase the activity of cannabidiolic acid synthase (CBDAS) and its expression levels in yeast, this study focused on the CBDAS^G183V-N482W mutant. Using computer-aided techniques and literature reviews, four mutation sites were further identified, resulting in the mutant CBDAS^{H114E-S116A-C176Y-G183V-N328Q-N482W}. The CBDAS gene was integrated into the Pichia pastoris genome via multiple transformation rounds, and relative enzyme activity was analyzed using high-performance liquid chromatography. The results of the molecular docking analysis revealed factors such as increased intermolecular forces, shorter bond lengths, and an increased number of amino acid–substrate interaction sites, which may have contributed to the enhanced catalytic activity of the mutant. The concentrations of CBDA and CBD produced by the CBDAS^{H114E-S116A-C176Y-G183V-N328Q-N482W} mutant were 71.543 ng/mL and 75.163 ng/mL, respectively, which were 11.87% and 11.53% greater than those produced by the CBDAS^G183V-N482W mutant. The recombinant CBDAS strain obtained after two consecutive transformations of the CBDAS^{H114E-S116A-C176Y-G183V-N328Q-N482W} vector presented the highest CBDAS expression levels and CBD and CBDA yields; compared with those obtained after a single transformation, the CBDA and CBD yields increased by 9.77% and 12.65%, respectively. In addition, the tolerance of the recombinant strain to induction culture conditions was analyzed, revealing that the strain could be induced to express the protein at temperatures ranging from 20 to 45 °C and at pH values ranging from 3 to 9, with optimal expression observed at 30 °C and pH 6. These findings provide theoretical and technical support for the production of enzyme preparations for the in vitro-directed biosynthesis of cannabidiol.

Cannabichromenic acid synthase (CBCAS) catalyzes a pivotal step in the biosynthesis of cannabichromene (CBC), and its activity level directly affects the production of CBC. In this study, a combined approach involving molecular docking and site-directed mutagenesis was employed to engineer the enzyme, with the goal of generating CBCAS variants that exhibit increased catalytic activity. The CBCAS gene was cloned from a cannabis cultivar with a high CBC content. The three-dimensional structure of the CBCAS protein was predicted using AlphaFold2. Molecular docking between the enzyme and its substrate, cannabigerolic acid (CBGA), was performed using Discovery Studio software. Mutation sites predicted to increase substrate binding affinity were identified, and the resulting recombinant plasmids were constructed and transformed into Pichia pastoris (P. pastoris) to generate engineered strains with increased CBCAS activity. Molecular docking analysis revealed that the CBCAS^Q106Y+E534R and CBCAS^Q124L+E534K double mutants exhibited increased noncovalent interactions and increased hydrophobic contact with the substrate, which likely accounted for their strengthened binding affinity for CBGA. Enzyme activity analysis revealed that compared with those of the wild type (CBCAS^WT), the enzymatic activities of CBCAS^Q106Y+E534R and CBCAS^Q124L+E534K were both significantly elevated (P < 0.05). These mutants catalyzed the conversion of CBGA to yield 93.3 ng/mL and 91.4 ng/mL CBC, representing increases of 37.5% and 34.7%, respectively, relative to CBCAS^WT. Heterologous expression of cannabis CBCAS was successfully achieved in P. pastoris, and mutant enzymes with increased catalytic activity were obtained, laying a foundation for the industrial production and application of recombinant CBCAS.

The AtVIP1-SbARF7 module controls root development via auxin signaling, offering insights into root regulatory networks and crop improvement. Plant root development is regulated by the auxin signaling pathway. It has been found that AtVIP1 (Arabidopsis thaliana VirE2-interacting Protein 1) gene overexpression promotes lateral root growth in sorghum, but its mechanism of action is not clear. The purpose of this study is to analyze the molecular mechanism by which AtVIP1 regulates sorghum root development through the auxin pathway, By constructing AtVIP1 overexpressing and silencing lines, it was found that overexpressing plants had a more developed root system (increased number of lateral root primordia and increased root volume), whereas silencing lines had suppressed growth. Expression analysis showed that AtVIP1 was enriched in root tips and lateral root primordia with subcellular localization in the nucleoplasm. Hormone treatment confirmed that AtVIP1 promoted auxin accumulation through the IAA pathway and stably up-regulated the expression of downstream genes such as SbYUCCA2 and SbIAA14. Y1H and EMSA confirmed that AtVIP1 directly binds to the SbARF7 promoter. The Dual-Luciferase Reporter constructed shows that AtVIP1 increases the promoter activity of SbARF7 by 2.3 times. After silencing SbARF7 in the background of AtVIP1-OE, the root branches decreased, indicating that AtVIP1 promotes root development by activating SbARF7 transcription. This study reveals the molecular mechanism by which the AtVIP1-SbARF7 module regulates root development through the auxin signaling pathway, providing new insights into the transcriptional regulatory network of root development and potential targets for the genetic improvement of crop root traits.

Fasciclin-like arabinogalactan proteins (FLAs) are crucial for plant growth and development. Utilizing whole genome data, this study delineated the number of genes, gene structure, chromosomal localization, protein structure, evolutionary relationships, and Gene Ontology (GO) annotations of the FLA family in sorghum (Sorghum bicolor L.). In addition, FLA genes’ expression in wild-type sorghum (P898012) under salt-alkali stress (SAS) was examined. We identified 26 FLA genes in sorghum. Phylogenetic analysis divided these genes into five subgroups, where members within the same subgroup exhibited extremely similar, though not identical, gene structures. A collinearity analysis of the sorghum FLA genes revealed that SbFLA19 does not share a homologous relationship with those in Zea and Arabidopsis, suggesting its uniqueness to sorghum. Promoter element analysis indicated that the FLA genes contain various response elements associated with abiotic stress. GO annotations demonstrated that most FLA proteins are primarily located on the plasma membrane and are involved in diverse biological processes. Transcriptomic data and qRT-PCR analysis under SAS revealed that members of the SbFLA family responded to stress at different times. These findings provide valuable references for breeding sorghum varieties tolerant to salt-alkali conditions.

4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) is widely used in the food industry as a spice and flavoring agent with high market demand. In this study, fructose-1,6-bisphosphate aldolase (FBA) and triose phosphate isomerase (TPI) were overexpressed in Zygosaccharomyces rouxii in the form of single and double genes, respectively, via electroporation. High-yield HDMF-engineered yeast strains were constructed by combining the analysis of gene expression levels obtained by real-time fluorescence quantitative PCR technology and HDMF production measured by HPLC. The results showed that there was a significant positive correlation between the production of HDMF and the expression levels of the FBA and TPI genes in yeast; the expression levels of the FBA and TPI genes were also positively correlated (p < 0.05). Compared with the wild type (WT), the engineered strains F10-D, T17-D, and TF15-A showed marked increases in HDMF production and FBA and TPI gene expression (p < 0.05) and exhibited great genetic stability with no obvious differences in biomass or colony morphology. In addition, the exogenous addition of d-fructose promoted the growth of Z. rouxii. Among the engineered strains, when fermented in YPD media supplemented with d-fructose for 5 days, TF15-A (overexpressing the FBA and TPI genes) generated the highest HDMF production of 13.39 mg/L, which is 1.91 times greater than that of the wild-type strain. The results above indicated that FBA and TPI, which are key enzymes involved in the process of HDMF biosynthesis by Z. rouxii, positively regulate the synthesis of HDMF at the transcriptional level. d-fructose can be used as a precursor for the biosynthesis of HDMF by engineered yeast in industrial production.

Key message SbMYC2 functions as a key regulator under JA signaling in enhancing drought tolerance of sorghum through direct activating SbGR1. Abstract Drought stress is one of the major threats to crop yield. In response to drought stress, functions of basic helix–loop–helix (bHLH) transcription factors (TFs) have been reported in Arabidopsis and rice, but little is known for sorghum. Here, we characterized the function of SbMYC2, a bHLH TF in sorghum, and found that SbMYC2 responded most significantly to PEG-simulated drought stress and JA treatments. Overexpression of SbMYC2 significantly enhanced drought tolerance in Arabidopsis, rice and sorghum. In addition, it reduced reactive oxygen species (ROS) accumulation and increased chlorophyll content in sorghum leaves. While silencing SbMYC2 by virus-induced gene silencing (VIGS) resulted in compromised drought tolerance of sorghum seedlings. Moreover, SbMYC2 can directly activate the expression of GLUTATHIONE-DISULFIDE REDUCTASE gene SbGR1. SbGR1 silencing led to significantly weakened drought tolerance of sorghum, and higher ROS accumulation and lower chlorophyll content in sorghum leaves were detected. In addition, SbMYC2 can interact with SbJAZs, suppressors of JA signaling, and thus can mediate JA signaling to activate SbGR1, thereby regulating sorghum's tolerance to drought stress. Overall, our findings demonstrate that bHLH TF SbMYC2 plays an important role in sorghum's response to drought stress, thus providing one theoretical basis for genetic enhancement of sorghum and even rice.

The quality and yields of Sorghum bicolor plants are seriously affected by saline-alkali conditions. NAC (NAM, ATAF, and CUC) transcription factors are plant specific and have various functions in plant development and response to various stresses. To investigate how GsNAC2 functions in sorghum responses to saline-alkali treatment, the characteristics of GsNAC2 were analysed by bioinformatics methods, and NaHCO3:Na2CO3 (5:1, 75 mM, pH 9.63) saline-alkali stress solution was applied when sorghum plants were 2 weeks old. The research results show that GsNAC2 belongs to the NAC gene family. GsNAC2 was significantly induced by saline-alkali treatment and strongly expressed in sorghum leaves. GsNAC2-overexpressing sorghum plants had increased plant height, dry weight, moisture content, root activity, leaf length, chlorophyll content, stomatal conductance, relative root activity, relative chlorophyll content, relative stomatal conductance, and relative transpiration rate after saline-alkali treatment. Lower H2O2 and O2− levels, relative permeability of the plasma membrane, and malondialdehyde (MDA) content were found in GsNAC2-overexpressing sorghum. In transcriptome analysis, clusters of orthologous groups (COG) analysis showed that a high proportion of differentially-expressed genes (DEGs) participated in defence mechanisms at each processing time, and 18 DEGs related to synthetic glutathione were obtained. Gene expression analysis revealed that key genes in glutathione biosynthesis pathways were upregulated. GR and GSH-Px activities were increased, and GSH accumulated more with the overexpression of GsNAC2 after saline-alkali treatment. Furthermore, these results suggest that GsNAC2 acts as a potentially important regulator in response to saline-alkali stress and may be used in molecular breeding to improve crop yields under adverse environmental conditions.