Lyase Gene Research Articles

In-depth insights into carbohydrate-active enzyme genes regarding the disparities in soil organic carbon after 12-year rotational cropping system field study

Carbohydrate-active enzymes (CAZymes) play a crucial role in plant-derived carbon utilization and decomposition and are influenced by the crop rotation system; however, our knowledge of how different agricultural systems impact CAZyme functionality is still limited. We conducted a metagenomic analysis to evaluate the functional genes of CAZymes in a 12-year in situ farmland with three commonly used crop rotation systems: wheat-maize rotation (WM), wheat-cotton rotation (WC), and wheat-soybean rotation (WS). We aimed to study the impact of long-term use of crop rotation, especially crop rotation involving soybean, on soil organic carbon (SOC) content and to gain an in-depth understanding of the CAZyme genes in context of the disparities in SOC. After 12 years, the SOC content was significantly higher in WS than in WC (5.44 %) and WM (17.6 %). Furthermore, the crop rotation system had a significant effect on the soil microbial communities and CAZyme function genes. Detailly, WS increased the phyla abundance of Proteobacteria, Actinobacteria, and Firmicutes and enriched the glycoside hydrolase (GH) and carbohydrate-binding modules (CBM) genes; WC increased the abundance of Acidobacteria and Bacteroidota and enriched the polysaccharide lyase gene; WM increased the abundance of Nitrospirae, Candidatus_Rokubacteria, Chloroflexi and Gemmatimonadetes and enriched the gene abundance of glycosyltransferases and auxiliary activity genes. Additionally, Acidobacteria, Proteobacteria, and Actinobacteria are key phyla involved in soil carbon cycling and collectively contribute >70 % of the total CAZyme functional genes, which highlights their importance. In addition, our results indicated that total nitrogen content played a major role in influencing genes related to CAZymes, especially those belonging to the GH family. Our study demonstrates that WS conferred the advantage of increasing SOC across the three crop rotation systems. CAZyme analysis revealed that WS's could potentially support the increased abundance of Proteobacteria, Actinobacteria and Firmicutes in the soil community, at the same time potentially leading to increased number of GH and CBM genes in the soil, which may bolster the decomposition and transformation of plant-derived carbon, thus promoting an increase in SOC content. The findings of this study offer new insights into the microbial factors contributing to SOC enhancement in rotation systems.

Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens strain ATCC 13124.

Clostridium perfringens, an opportunistic pathogen, produces mu-toxin hyaluronidases including endo-β-N-acetylglucosaminidases (Nags) as a virulence invasion factor. To clarify an intrinsic factor for degradation of host extracellular hyaluronan, we focused on hyaluronate lyase (HysA), distinct from endo-β-N-acetylglucosaminidases. C. perfringens strain ATCC 13124 was found to assimilate host-derived extracellular mucosubstances, hyaluronan and mucin, which induced expression of the hyaluronan-related genetic cluster, including hyaluronate lyase gene (hysA), but repressed endo-β-N-acetylglucosaminidase genes. This genetic cluster is conserved in some strains of C. perfringens. The recombinant strain ATCC 13124 hyaluronate lyase HysA showed an hyaluronan-degrading activity through β-elimination reaction. The hyaluronan-degrading enzyme in the culture supernatant of strain ATCC 13124 exhibited the lyase activity and was identical to the recombinant hyaluronate lyase on the native-PAGE gel, followed by activity straining. These results demonstrated that the intrinsic hyaluronan-degrading enzyme of C. perfringens strain ATCC 13124 is hyaluronate lyase HysA, but not hyaluronidases NagH, NagJ, and NagK.

Dynamic Change of Volatile Fatty Acid Derivatives (VFADs) and Their Related Genes Analysis during Innovative Black Tea Processing

Volatile fatty acid derivatives (VFADs) play a significant role in contributing to flowery–fruity flavor black tea. Innovative black tea is typically crafted from aroma-intensive tea cultivars, such as Jinmudan, using defined production methodologies. In this study, the during-processing tea leaves of innovative black tea were applied as materials, and we selected a total of 45 VFADs, comprising 11 derived aldehydes, nine derived alcohols, and 25 derived esters. Furthermore, the dynamic variations of these VFADs were uncovered. Transcriptome analysis was performed to identify genes involved in the LOX (lipoxygenase) pathway, resulting in the identification of 17 CsLOX genes, one hydrogen peroxide lyase (CsHPL) gene, 11 alcohol dehydrogenases (CsADH) genes, 11 genes as acyl CoA oxidase (CsACOX) genes, and three allene oxide synthase (CsAOS) genes. Additionally, the expression levels of these genes were measured, indicating that the processing treatments of innovative black tea, particularly turn-over and fermentation, had a stimulation effect on most genes. Finally, qRT-PCR verification and correlation analysis were conducted to explain the relationship between VFADs and candidate genes. This study aims to provide a reference for illuminating the formation mechanisms of aroma compounds in innovative black tea, thereby inspiring the optimization of innovative processing techniques and enhancing the overall quality of black tea.

The role of ATP citrate lyase in myelin formation and maintenance.

Formation of myelin by Schwann cells is tightly coupled to peripheral nervous system development and is important for neuronal function and long-term maintenance. Perturbation of myelin causes a number of specific disorders that are among the most prevalent diseases affecting the nervous system. Schwann cells synthesize myelin lipids de novo rather than relying on uptake of circulating lipids, yet one unresolved matter is how acetyl CoA, a central metabolite in lipid formation is generated during myelin formation and maintenance. Recent studies have shown that glucose-derived acetyl CoA itself is not required for myelination. However, the importance of mitochondrially-derived acetyl CoA has never been tested for myelination in vivo. Therefore, we have developed a Schwann cell-specific knockout of the ATP citrate lyase (Acly) gene to determine the importance of mitochondrial metabolism to supply acetyl CoA in nerve development. Intriguingly, the ACLY pathway is important for myelin maintenance rather than myelin formation. In addition, ACLY is required to maintain expression of a myelin-associated gene program and to inhibit activation of the latent Schwann cell injury program.

Roles of Three FgPel Genes in the Development and Pathogenicity Regulation of Fusarium graminearum.

Fusarium head blight (FHB) is a devastating fungal disease caused by Fusarium graminearum. Pectin lyase, a pectinase, acts on the α-1,4-glycosidic linkage of galacturonic acid primarily by β-elimination. In this study, three pectin lyase genes (FgPel1, 2, 3) in F. graminearum were selected, and deletion mutants (ΔFgPel1, 2, 3) were constructed by homologous recombination for functional characterization. The gene deletions affected the morphology and growth rate of F. graminearum on pectin medium at various concentrations, with the growth rate of ΔFgPel1 being more significant. The growth of ΔFgPel1 colonies slowed at pH 4, with optimal growth at pH 6.5, whereas ΔFgPel2 and ΔFgPel3 exhibited greater inhibition at pH 8. Colony morphology and diameter of the deletion mutants showed no significant differences compared to the wild-type strain PH-1, and there was no effect on conidial production or germination rate. Pathogenicity assays demonstrated that gene deletion significantly reduced the ability of F. graminearum to infest corn silks and wheat ears, and that ΔFgPel2 showed a more pronounced reduction in pathogenicity on wheat spikes. In summary, the pectin lyase genes (FgPel1, 2, 3) are involved in pectin utilization and are influenced by external pH conditions, which attenuate the pathogenicity of F. graminearum without affecting its vegetative growth or asexual spore formation. These findings elucidate the roles of these genes and provide a basis for controlling FHB.

Impacts of Selenium Supplementation on Soil Mercury Speciation, Soil Properties and Mercury-Resistant Microorganisms and Resistant Genes

Soil mercury (Hg) contamination is a serious threat to local ecology and public health. Exogenous selenium (Se) supplementation can effectively reduce the toxicity of Hg. However, the mechanisms affecting the changes in soil Hg speciation, soil properties and the microbial Hg-resistant system during the Se–Hg interaction after exogenous Se supplementation are not clear. Therefore, in this study, soil culture experiments were conducted to analyze the effects of different Se additions on the transformation of Hg speciation, soil properties and Hg-resistant microorganisms and resistant genes (mer operon). The results indicated that Se supplementation facilitated the transformation of soil Hg from bioavailable (exchangeable and carbonate-bound) to stable forms (organic material-bound and residual), significantly reducing Hg bioavailability. Se supplementation notably decreased the electrical conductivity of Hg-contaminated soil, but had no significant effect on the soil pH, organic matter content, cation exchange capacity or alkaline phosphatase and catalase activities. The maximum activity levels of soil sucrase and urease were observed when 1 mg kg−1 Se was added. Se significantly inhibited soil peroxidase and ascorbate oxidase activities, thereby alleviating the oxidative stress in the soil system caused by Hg. Additionally, Se significantly activated the Hg-resistant system in soil microorganisms by either decreasing or increasing the regulatory genes merD and merR, and it significantly upregulated the cytoplasmic protein gene merP and the membrane protein genes merC, merF and merT. This further increased the abundance of the organomercury lyase gene merB and the mercuric reductase gene merA, promoting the conversion of Hg species to Hg⁰. Furthermore, the abundance of mer operon-containing microorganisms, such as Thiobacillus ferrooxidants, Pseudomonas, Streptomyces and Cryptococcus, significantly increased with Se addition, explaining the role of soil microorganisms in mitigating soil Hg stress via Se supplementation.

An S-methyltransferase that produces the climate-active gas dimethylsulfide is widespread across diverse marine bacteria.

Hydrogen sulfide (H2S), methanethiol (MeSH) and dimethylsulfide (DMS) are abundant sulfur gases with roles in biogeochemical cycling, chemotaxis and/or climate regulation. Catabolism of the marine osmolyte dimethylsulfoniopropionate (DMSP) is a major source of DMS and MeSH, but both also result from S-methylation of H2S via MddA, an H2S and MeSH S-methyltransferase whose gene is abundant in soil but scarce in marine environments. Here we identify the S-adenosine methionine (SAM)-dependent MeSH and H2S S-methyltransferase 'MddH', which is widespread in diverse marine bacteria and some freshwater and soil bacteria. mddH is predicted in up to ~5% and ~15% of seawater and coastal sediment bacteria, respectively, which is considerably higher than mddA. Furthermore, marine mddH transcript levels are similar to those for the most abundant DMSP lyase gene dddP. This study implies that the importance of H2S and MeSH S-methylation pathways in marine environments is significantly underestimated.

Overexpression of sesame O-acetylserine(thiol)lyase induces male sterility by delaying tapetum programmed cell death

Male sterile lines are key resources for hybrid seed production and for ensuring high varietal purity. However, the genes and mechanisms underlying sesame male sterility remain largely unknown. Hence, this study identified an O-acetylserine(thiol)lyase gene SiOASTL1 and functionally characterized its roles in inducing defective anther development. Spatiotemporal expression analysis revealed that SiOASTL1 is significantly (2.7 fold) up-regulated in sterile sesame anthers at the microspore stage compared with fertile ones. Sequence and phylogenetic analyses showed that SiOASTL1 is homologous to Arabidopsis OAS-TL plastid isoforms. We thus overexpressed SiOASTL1 in Arabidopsis to unravel its regulatory roles. Cytological observation revealed that SiOASTL1 overexpression transformed transgenic plants into male sterile lines arising at the microspore development stage. SiOASTL1 overexpression decreased cysteine biosynthesis and down-regulated the expression of the sporopollenin synthesis-related genes, including AtTKPR1, AtTKPR2, AtPKSA, and AtPKSB in transgenic Arabidopsis. Consequently, the tapetum programmed cell death (PCD) was delayed, resulting in the formation of defective pollen grains with irregular walls and empty cytoplasm. Our findings prove that the induction of SiOASTL1 expression disrupts pollen development and contributes to sesame male sterility. Moreover, these results suggest that genetic manipulation of SiOASTL1 expression may facilitate the development of new hybrid varieties in sesame and other crops.

Cold plasma treatment delays the texture softening of apricot fruit through PAPL-PL-pectin changes

Apricot, a forest fruit with distinct characteristics, has witnessed a continuous expansion of its market scale over the years. However, the high loss rate caused by fruit texture softening poses a significant challenge to the development of the apricot industry. The effect of cold plasma treatment on the texture was investigated. The findings revealed that compared to the control group, the firmness of apricot fruit increased by 49%, 31%, and 30% after treatment for 20, 40, and 60 s, respectively. Applying an optimal cold plasma treatment (20–40 s) can effectively inhibit the degradation of pectin component branches, thereby delaying the increase in water-soluble pectin (WSP) and iron - soluble pectin (ISP) content, reducing the decline in covalent - soluble pectin (CSP) content, ultimately enhancing fruit firmness and extending the storage period of apricot. RNA-seq results revealed the involvement of pectin decomposition mediated by pectin lyase (PL) in the process of texture softening of apricot fruit, with a key role played by the pectin lyase gene (PaPL). These findings not only enrich the mechanism of texture softening of apricot fruit, but also provide valuable insights for utilizing cold plasma in post-harvest preservation of apricot fruit.

D53 represses rice blast resistance by directly targeting phenylalanine ammonia lyases.

In rice, DWARF 53 directly binds to the promoters of seven phenylalanine ammonia lyase genes, OsPAL1˜OsPAL7, and represses their expression, leading to decreased lignin accumulation and compromised resistance against Magnaporthe oryzae.

Genome-wide identification of the pectate lyase (PEL) gene family members in Malvaceae, and their contribution to cotton fiber quality

Pectin is a major constituent of the plant cell wall. Pectate lyase (PEL, EC 4.2.2.2) uses anti-β-elimination chemistry to cleave the α-1,4 glycosidic linkage in the homogalacturonan region of pectin. However, limited information is available on the comprehensive and evolutionary analysis of PELs in the Malvaceae. In this study, we identified 597 PEL genes from 10 Malvaceae species. Phylogenetic and motif analyses revealed that these PELs are classified into six subfamilies: Clades I, II, III, IV, Va, and Vb. The two largest subfamilies, Clades I and II, contained 237 and 222 PEL members, respectively. The members of Clades Va and Vb only contained four or five motifs, far fewer than the other subfamilies. Gene duplication analysis showed that segmental duplication played a crucial role in the expansion of the PEL gene family in Gossypium species. The PELs from Clades I, IV, Va, and Vb were expressed during the fiber elongation stage, but nearly all PEL genes from Clades II and III showed no expression in any of the investigated fiber developmental stages. We further performed single-gene haplotype association analysis in 2,001 G. hirsutum accessions and 229 G. barbadense accessions. Interestingly, 14 PELs were significantly associated with fiber length and strength traits in G. barbadense with superior fiber quality, while only eight GhPEL genes were found to be significantly associated with fiber quality traits in G. hirsutum. Our findings provide important information for further evolutionary and functional research on the PEL gene family members and their potential use for fiber quality improvement in cotton.

Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

Molecular mechanism of delayed development by interfering RNA targeting the phenylalanine ammonia lyase gene (pal1) in Pleurotus ostreatus

The blockage of the development of edible mushrooms will affect the production cycle and yield of fruiting bodies. Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is an enzyme that catalyses the deamination of phenylalanine to form trans-cinnamic acid. Previous results have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus. Herein, we used wild type (WT) and RNA interference (RNAi) strains to study the molecular regulation of pal1 by RNA sequencing and agrobacterium-mediated genetic transformation. Our results showed that interference with the pal1 gene resulted in a decrease in the total PAL enzyme activity and the total phenol content, as well as an increase in the intracellular H2O2 content. RNA-Seq data demonstrated that the significantly enrichment KEGG terms were mainly related to the peroxisome pathway, MAPK signalling pathway-yeast and three other pathways, moreover, the catalase gene cat1 involved in multiple pathways that were enriched above. Exogenous H2O2 significantly enhanced the transcription of the cat1 gene and elevated total CAT enzyme activity. Moreover, the levels of cat1 gene transcription and the total CAT enzyme activity in the RNAi-pal1 strains were gradually closed to those in the WT strain through the removal of H2O2, which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H2O2 content. Finally, the overexpression of the cat1 gene in P. ostreatus caused growth retardation, especially during the process of primordia formation. In conclusion, this study demonstrated that PAL1 affects cat1 gene expression through the signalling molecule H2O2 and regulates the development of P. ostreatus. Our findings have enhanced the understanding of the molecular developmental mechanism of edible mushrooms.

Genome-Wide Identification of the Pectate Lyase Gene Family in Potato and Expression Analysis under Salt Stress.

Pectin is a structural polysaccharide and a major component of plant cell walls. Pectate lyases are a class of enzymes that degrade demethylated pectin by cleaving the α-1,4-glycosidic bond, and they play an important role in plant growth and development. Currently, little is known about the PL gene family members and their involvement in salt stress in potato. In this study, we utilized bioinformatics to identify members of the potato pectate lyase gene family and analyzed their gene and amino acid sequence characteristics. The results showed that a total of 27 members of the pectate lyase gene family were identified in potato. Phylogenetic tree analysis revealed that these genes were divided into eight groups. Analysis of their promoters indicated that several members' promoter regions contained a significant number of hormone and stress response elements. Further, we found that several members responded positively to salt treatment under single salt and mixed salt stress. Since StPL18 exhibited a consistent expression pattern under both single and mixed salt stress conditions, its subcellular localization was determined. The results indicated that StPL18 is localized in the endoplasmic reticulum membrane. The results will establish a foundation for analyzing the functions of potato pectate lyase family members and their expression under salt stress.

Novel insights into dimethylsulfoniopropionate cleavage by deep subseafloor fungi

Dimethylsulfoniopropionate (DMSP), a key organic sulfur compound in marine and subseafloor sediments, is degraded by phytoplankton and bacteria, resulting in the release of the climate-active volatile gas dimethylsulfide (DMS). However, it remains unclear if dominant eukaryotic fungi in subseafloor sediments possess specific abilities and metabolic mechanisms for DMSP degradation and DMS formation. Our study provides the first evidence that fungi from coal-bearing sediments ∼2 km below the seafloor, such as Aspergillus spp., Chaetomium globosum, Cladosporium sphaerospermum, and Penicillium funiculosum, can degrade DMSP and produce DMS. In Aspergillus sydowii 29R-4-F02, which exhibited the highest DMSP-dependent DMS production rate (16.95 pmol/μg protein/min), two DMSP lyase genes, dddP and dddW, were identified. Remarkably, the dddW gene, previously observed only in bacteria, was found to be crucial for fungal DMSP cleavage. These findings not only extend the list of fungi capable of degrading DMSP, but also enhance our understanding of DMSP lyase diversity and the role of fungi in DMSP decomposition in subseafloor sedimentary ecosystems.

Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

Genome Sequence Resource of a Taro Bacterial Soft Rot Pathogen, Dickeya fangzhongdai ZXC1

Dickeya fangzhongdai ZXC1 is a newly identified highly virulent causal agent of taro bacterial soft rot disease isolated from a taro sample showing typical soft rot symptoms in Shaoguan, Guangdong Province, China. The pathogen produces plant cell wall–degrading enzymes that macerate the taro tube tissues. In this study, we report the whole-genome sequencing analysis of strain ZXC1. The results showed that strain ZXC1 has one circular DNA chromosome of 5,129,951 bp with a 56.59% G+C content. In addition to sharing a conserved zms gene cluster that encodes the genes for biosynthesis of phytotoxin zeamines and 11 copies of predicted pectate lyase genes, the strain ZXC1 genome contains more prophage loci and a higher number of type IV secretion system and type IV secretion system gene clusters than the D. fangzhongdai strains isolated from pears or phalaenopsis, which may account for its strong virulence phenotype. The data from this study present a valuable resource for elucidation of the pathogenic mechanisms of D. fangzhongdai and may aid in developing new disease control approaches to safeguard taro production. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Pectate lyase genes from Radopholus similis and their application in pathotype identification

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes.Key points• Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed.• The expression of Rs-pels is different in two pathotypes of Radopholus similis.• A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Fed-batch fermentation of Mucor circinelloides reveals significant improvement in biomass and lipid accumulation through performance evaluation, chemical analysis, and expression profiling

This study aimed to improve the lipid and biomass yields of Mucor circinelloides WJ11 by implementing four different fed-batch fermentation strategies, varied in time and glucose concentration (S1-S4). The S1 fermentation strategy yielded the highest biomass, lipid, and fatty acid content (22 ± 0.7 g/L, 53 ± 1.2 %, and 28 ± 1.6 %) after 120 and 144 h, respectively. The γ-linolenic acid titer of 0.75 ± 0.0 g/L was greatest in S3 after 48 h. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the transcription of key genes involved in lipid accumulation. The glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase genes showed increased expression levels. Fourier-transform infrared (FTIR) spectroscopy was used to analyze the biochemical profile during fermentation strategies. Optimal abiotic factors for production efficiency included pH 6.5, 25–26 °C, 15 % (v/v) inoculum, 500 rpm, 20 %-30 % dissolved oxygen, and 120 h fermentation. Glucose co-feeding offers valuable insights to develop effective fermentation strategies for lipid production.