Quercetin is one of the main medicinal ingredients of Euphorbia maculata, and chalcone synthase（CHS）is one of key enzymes in the biosynthesis of quercetin. To explore the mechanism of quercetin biosynthesis in E. maculata, a CHS gene named EmCHS（GenBank access number: ON652865） was cloned from E. maculata based on transcriptome sequencing results, combined with technologies of 3’RACE and Tail-PCR. The amino acid sequence was aligned, and physicochemical properties, transmembrane region, subcellular localization and phylogenetic relationships of EmCHS protein were analyzed. And the expressions of EmCHS in different tissues during different growth stages were detected with RT-qPCR. The results showed that the ORF of EmCHS gene was 1 194 bp, encoding 397 amino acids. The EmCHS protein was located in the cytoplasm. The theoretical isoelectric point of the protein was 5.96 and the theoretical molecular weight was 43.48 kD,and it did not contain a transmembrane region and was a hydrophilic protein with stable structure. The results of amino acid sequence alignment and phylogenetic relationships analysis revealed that EmCHS had the highest similarity（91.92%）with the amino acid sequence of Manihot esculenta in Euphorbiaceae family, which was consistent with the characteristics of plant taxonomy. The analysis of RT-qPCR confirmed that EmCHS gene was expressed in different tissues during different growth stages, and there were significant differences. The expression of EmCHS gene was the lowest in the leaf at the reproduction stage, while the highest in the root at the reproduction stage. Besides, the promoter（GenBank access number: OP626754）length of EmCHS gene cloned was 971 bp, including TATA-box, CAAT-box, MYB and MYC transcription factor binding sites, multiple light response and hormone response cis-acting elements, and so on. These results will provide a foundation for further research on gene function and expression regulation of EmCHS gene.
Lactate dehydrogenase(LDH)catalyzes the reversible conversion between pyruvate and lactic acid.In order to explore the role of LDH genes in heat stress response of Pleurotus ostreatus,eight coding genes of LDH were identified in the genome of P.ostreatus strain CCMSSC00389.The bioinformatics analyses showed that three genes encoded D-lactate dehydrogenase and five gene encoded L-lactate dehydrogenase.The results from the amino acid sequence alignment,phylogenetic analysis,three-dimensional structure and subcellular localization showed that three D-lactate dehydrogenases sharing a high homolog,which belonged to cytochrome C-dependent D-lactate dehydrogenase,and D-LDH2 and D-LDH3 were located in mitochondria.The five L-lactate dehydrogenases having a low homolog were divided into three different types according to electron acceptor and subcellular localization.The gene transcription changes of lactate dehydrogenase at 36℃and 40℃indicated the similar expression characteristics shared by D-ldh3,L-ldh5 and L-ldh7,as well as by L-ldh6 and L-ldh8;while the distinctive expression characteristics within D-ldh1,D-ldh2 and L-ldh4 was observed,which revealed varied response ways of the LDH genes to different stress temperatures.The total lactate dehydrogenase enzyme activity decreased and the intracellular lactic acid content increased under the heat stress.The inhibiting effects of lactate dehydrogenase inhibitor and exogenous lactic acid on the mycelia growth of P.ostreatus suggested that LDH regulated the mycelia growth under heat stress.It is speculated that P.ostreatus lactate dehydrogenase affects the conversion of intracellular LDH to pyruvate by reducing gene transcription level and enzyme activity,resulting in excessive accumulation of intracellular lactic acid and cell damage,and finally inhibits the growth rate of hyphae.
Cellulosic ethanol,as a clean and renewable green energy,has promising application prospects.However,the fermentation process of Saccharomyces cerevisiae using lignocellulose to produce ethanol is susceptible to various stresses,thus improving the stress tolerance of fermenting microorganisms has become an essential topic in the field of biorefinery.In this work,we attempted to engineer cells to use the output of the internal redox-sensitive biosensor Yap1 to drive intracellular metabolic reactions(including ROS metabolism and aldehyde degradation),which will improve the tolerance of S.cerevisiae to reactive oxygen species and aldehyde inhibitors.Firstly,we analyzed the sequences of several native endogenous promoters(PTRR1,PTRX2,and PMET16)in S.cerevisiae that can be regulated by the redox-sensitive biosensor Yapl and their response strengths to typical inhibitors in lignocellulosic hydrolysates.Secondly,we designed redox-sensitive genetic parts in order to improve the stress resistance of S.cerevisiae by combining the promoters with corresponding resistance genes.Finally,we tried to integrate the better performing GP-CTT and GP-ADH in tandem to construct a dual-genetic parts system,which was named GP-AC.The integration greatly improved the ability of S.cerevisiae to cope with multiple complex stresses,and the cell death rate decreased by 69.6%under the coexistence of aldehyde and oxidation stress.Compared with GP-CTT,the specific growth rate,glucose consumption rate and ethanol production rate of GP-AC increased by 64.2%,60.1%,and 58.9%,respectively.At the same time,the catalase activity of the recombinant strain increased by 40.2%compared with the single genetic part.In conclusion,this study systematically improved the stress tolerance of S.cerevisiae by rationally designing the genetic circuit of redox-sensitive genetic parts and strengthening the intracellular vital antioxidant enzymes and aldehyde degradation pathways.It provides new insights for rationally designing and constructing feedback genetic circuits to dynamically improve yeast robustness.
Lignocellulosic biomass(LCB)is a promising alternative to fossil material,the biofuels,biochemicals and biomaterials are produced via its biorefinery,which may reduce carbon emissions,contributing to achieve the carbon peaking and carbon neutrality goals.Therefore,LCB is receiving more and more attentions.However,there are multiple steps involved in lignocellulosic biorefinery including pretreatment,microbial fermentation and product purification,in which,various compounds generated from pretreatment of LCB inhibit cell growth and fermentation performance of microbes,which is one of the bottlenecks of bioconversion efficiency.Escherichia coli is a commonly used host for lignocellulosic biorefinery and extensively used for the production of many compounds,thus,it is of great importance to study the stress tolerance of E.coli to inhibitors in lignocellulosic hydrolysates for improving lignocellulosic biorefinery efficiency.This paper first introduced the main components and structures of lignocellulose,and briefly elucidated pretreatment methods to lignocellulose as well as main inhibitors in the hydrolysate after pretreatment.Then the paper summarized the toxic effects of main inhibitor furans,carboxylic acids and phenolics in the hydrolysate of lignocellulose on E.coli,as well as the mechanisms of stress tolerance against these inhibitors and the engineering targets for improving strain tolerance based on the mechanisms.Finally,the paper reviewed the strategies for strain engineering to improve the tolerance of E.coli to above motioned inhibitors,including random mutagenesis,adaptive laboratory evolution and omics-assisted rational design,,aiming to provide references for metabolic engineering of efficient E.coli strains for lignocellulosic biorefinery.
Mediator complex,as an essential coactivator in the eukaryotic transcription machinery,plays a crucial role in bridging RNA polymerase Ⅱ and various transcription factors to regulate gene transcription.When plants encounter various abiotic stresses,the mediator complex integrates stress signals and activates or suppresses the expressions of target genes,thereby helping plants to adapt to environmental stress.This article summarizes the discovery of the mediator complex,and different functions of plant mediator complex under drought,salt,and extreme temperature conditions.It also discusses future research directions of the mediator complex to gain a deeper understanding of its role in regulating responses to abiotic stress.
