MVA Pathway Research Articles

Efficient biosynthesis of β-caryophyllene in Saccharomyces cerevisiae by β-caryophyllene synthase from Artemisia argyi

Artemisia argyi H. Lév. & Vaniot is an important traditional Chinese medicinal plant known for its volatile oils, which are the main active components of A. argyi, including monoterpenes, sesquiterpenes and their derivatives. Despite its medicinal significance, the biosynthesis of sesquiterpenoids in A. argyi remains underexplored. In this study, we identified four β-caryophyllene synthases from A. argyi. A high-yield β-caryophyllene engineered Saccharomyces cerevisiae cell factory has been built in this study. By fusing ERG20 and AarTPS88 with a flexible linker (GGGS)2 and enhancing metabolic flux in the MVA pathway (HIF-1, tHMGR, and UPC2-1), we achieved a titer of β-caryophyllene reached 15.6 g/L by fed-batch fermentation in a 5 L bioreactor. To our knowledge, this represents the highest reported titer of β-caryophyllene in yeast to date. This study provides a valuable tool for the industrial-scale production of β-caryophyllene.

Enhancement of Sesquiterpenoid Production by Methyl Jasmonate in Atractylodes chinensis Adventitious Root Culture and its Transcriptional Regulation.

The adventitious root (AR) culture of Atractylodes chinensis is an efficient platform for sustainable production of its sesquiterpenoid compounds (atractylon and β-eudesmol). However, their limited accumulation levels need an effective elicitation approach, and the present study solved this problem using methyl jasmonate (MeJA) as an elicitor. The effects of its treatment concentration and duration on metabolite production were investigated. The ARs treated with 100 µM MeJA for seven days increased atractylon and β-eudesmol by 3.64- and 1.90-fold, respectively, compared with the control. This study further performed transcriptome analysis to explore the transcriptional regulation mechanism of the MeJA elicitation. A total of 124,464 unigenes were identified in A. chinensis ARs, of which 3,568 genes were upregulated and 3,864 genes were downregulated under the MeJA treatment. The MeJA treatment activated the endogenous JA biosynthesis and signaling pathways and sesquiterpenoid biosynthesis. The MeJA treatment more significantly activated the MEP pathway than the MVA pathway. In addition, 14 genes encoding terpene synthase were identified to be significantly upregulated. A total of 2,700 transcription factors (TFs) were identified in A. chinensis ARs, of which Tify, MYB, and MADS were significantly enriched under the MeJA treatment. We predicted a new antagonistic interaction between MYC2 and CPP TFs, which was significantly regulated by the MeJA treatment. The results of real-time quantitative PCR and enzyme activity assays proved the reliability of the transcriptome data. This study will help improve the in vitro production system of A. chinensis sesquiterpenoids and understand the transcriptional regulation mechanism of MeJA elicitation.

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies

BackgroundLimonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.ResultsIn this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.ConclusionsIn this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Synthesis of myrcene using a hybrid cell-free reaction system

Myrcene is a typical monoterpene with applications as pharmaceutical product, biofuel, and so forth. However, only traces of myrcene accumulate in plants, and in vivo production of monoterpene in microbial cells also contains multiple restrictive factors. To alleviate this limitation, a hybrid cell-free metabolic engineering (CFME) system was developed in this study and a series of CFME strategies were employed for the engineering of myrcene synthase from glucose. First, a convenient CFME system was established, in which Pichia pastoris and Escherichia coli lysates enriched with MVA pathway and myrcene synthase respectively were used as “plug and play” modules and combined. After optimization, the myrcene titer was significantly improved by 119.26%. Second, rational design of myrcene synthase was performed by conserved residues modification and substrate tunnel engineering. Myrcene synthase variant N238S outperformed the wild-type enzyme in CFME system, and the myrcene titer value reached 56.80 mg/L. Molecular dynamics simulations provided significant mechanistic insights into the enhanced catalytic capability and myrcene production. This work highlights the feasibility of hybrid CFME system to connect tandem module and sustain complex metabolism for myrcene biomanufacturer. It also illustrates the value of the cell-free approach as a starting point for the rational engineering of monoterpene synthase.

Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product β-ionone was produced at a titer of 0.87 mg/L by expressing the β-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Comparative Transcriptomics Analysis Reveals Rusty Grain Beetle's Aggregation Pheromone Biosynthesis Mechanism in Response to Starvation.

Pheromones are the basis of insect aggregation, mating, and other behaviors. Cucujoid grain beetles produce macrocyclic lactones as aggregation pheromones, yet research on their biosynthesis at the molecular level remains limited. The rusty grain beetle, C. ferrugineus, is an important economic species in China. Although two aggregation pheromone components have been identified, their suspected biosynthesis via the MVA pathway and the FAS pathway lacks molecular elucidation. Previous evidence supports that starvation affects the production of aggregation pheromones. Therefore, we constructed comparative transcriptome libraries of pheromone production sites in C. ferrugineus under starvation stress and identified genes related to pheromone biosynthesis and hormone regulation. A total of 2665 genes were significantly differentially expressed, of which 2029 genes were down-regulated in starved beetles. Putative C. ferrugineus genes directly involved in pheromone biosynthesis were identified, as well as some genes related to the juvenile hormone (JH) pathway and the insulin pathway, both of which were depressed in the starved beetles, suggesting possible functions in pheromone biosynthesis and regulation. The identification of genes involved in macrolide lactone biosynthesis in vivo holds great significance, aiding in the elucidation of the synthesis and regulatory mechanisms of cucujoid grain beetle pheromones.

Combined analysis of GC-MS, RNA-seq and ATAC-seq elucidates the essential oils variation and terpenes biosynthesis in Citrus grandis ‘Tomentosa’

Citrus grandis ‘Tomentosa’ (CGT) contain abundant essential oils (EOs), while the variation of them among different varieties is still unclear which hinders the fully utilization of CGT EOs in industry. Besides, the molecular mechanism underlying the accumulation of EOs in Citrus family remains largely unknown. In this study, multi-omics including GC-MS (gas chromatography-mass spectrometry), RNA-seq (ribonucleic acid sequencing), and ATAC-seq (assay for transposase-accessible chromatin using sequencing) were applied for three varieties of CGT (‘ZM’, ‘FM’, and ‘GQ’) to reveal the CGT EOs variation at the chemical, transcriptional and chromatinic levels. CGT EOs mainly consist of monoterpenes and sesquiterpenes, and the total terpenes amounts in three varieties rank as ‘ZM’ > ‘FM’ > ‘GQ’. The total amount of monoterpenes in ‘ZM’ is almost triple than that in ‘FM’ and is septuple than that in ‘GQ’. The average contents of sesquiterpenes in the EOs of ‘ZM’, ‘FM’, and ‘GQ’ are 73.9 ug/g, 42.65 ug/g and 1.75 ug/g, respectively. RNA-seq analysis among the three varieties demonstrated that the structural genes of 1-DEOXY-D-XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) and 1-DEOXY-D-XYLYLOSE 5-PHOSPHATE REDUCTOISOMERASE (DXR) are differentially expressed genes (DEGs) in MEP pathway for the biosynthesis of monoterpenes, and 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE (HMGR) is the DEG for sesquiterpenes synthesis in MVA pathway. Integrated GC-MS, RNA-seq and ATAC-seq analysis reveals 9 transcriptional factors were predicted to regulate the terpenes synthesis in the exocarps of CGT. The present investigation provides important information for manufacturers and breeders of CGT who require raw materials and germplasm of CGT in better quality for the production of EOs.

Heterologous biosynthesis of prenylated resveratrol through multiplex metabolic engineering in Escherichia coli

2-C-Prenyl resveratrol was successfully produced by reconstructing the MVA pathway, resveratrol synthesis pathway and metabolic flux engineering to accumulate acetyl-CoA and malonyl-CoA.

Transcriptome analysis of the mechanism of endophytic fungus CHS3 promoting saikosaponin d synthesis in Bupleurum scorzonerifolium Willd. suspension cells

Saikosaponin d (SSd) is the main component of Bupleuri Radix, a famous traditional Chinese herbal medicine, with high medicinal value. An endophytic fungus (CHS3) was isolated from Bupleurum scorzonerifolium Willd. in the early stage of our research, and we found that CHS3 could promote the accumulation of SSd in Bupleurum scorzonerifolium Willd. suspension cells (BSS cells). It is of practical significance to identify the mechanism that CHS3 promoted the accumulation of SSd and increased the production of SSd in suspension cells. To search the influence of CHS3 on SSd synthesis in the BSS cells, we co-cultured CHS3 with the BSS cells and compared the SSd content in BSS cells before and after co-culture using high-performance liquid chromatography (HPLC). Then the Illumina HiSeq 2500 was performed to detect the transcriptome of the BSS cells before and after co-culture and analyzed for the KEGG enrichment. The expression of genes involved in SSd synthesis was finally corroborated by qPCR analysis. Among which 11 key genes in connection with SSd synthesis were increased in BSS cells of co-cultured group compared with the BSS cells of the control group. In conclusion, CHS3 could promote the accumulation of SSd in BSS cells, and the molecular mechanism was related to its ability to regulate the MVA pathway, the calcium signaling pathway, and the AMPK signaling pathway by upregulating the expressions of ANT, CypD, CaM, AMPK, AATC, HMGS, HMGR, MVK, MVD, SS, and SE.

Engineering Yarrowia lipolytica for the biosynthesis of geraniol

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.

Metabolomic and transcriptomic integrated analysis revealed the decrease of monoterpenes accumulation in table grapes during long time low temperature storage

Low temperature is the commonly used technique for maintaining the quality of table grapes during postharvest storage. However, this technique could strongly affect the aromatic flavor of fruit. Monoterpenes are the key compounds contributing to the Muscat aromas of grapes. The detailed information and molecular mechanisms underlying the changes in monoterpenes during postharvest low temperature storage have not been thoroughly characterized. In this study, the effects of low temperature storage on the free and bound monoterpene profiles in four cultivars of table grape were determined at both the transcriptomic and metabolomic levels. A total of 27 compounds in both free and bound forms were identified in the four cultivars and showed quantitative differences between the cultivars. Hierarchical cluster and principal component analysis indicated that the free and bound monoterpene profiles were remarkably affected by the low temperature storage. The monoterpenes in the same biosynthesis pathway were clustered together and showed similar evolution trends during low temperature storage. And the content of most of free monoterpenes underwent a rapid decline during low-temperature storage at a certain stage, but the time was different in 4 grape cultivars. Transcriptomic analysis revealed that the expression of DXS, HDR, GPPS and TPS genes involved in the monoterpene synthesis pathway were consistent with the changes in the accumulation of monoterpene compounds. While the expression of HMGS, HMGR genes in MVA pathway and branch genes GGPPS and FPPS were negatively correlated with the accumulation of monoterpenes. The findings provide new insights into the underlying mechanisms of the berry aroma flavor change during low temperature storage.

Genome analysis of a halophilic Virgibacillus halodenitrificans ASH15 revealed salt adaptation, plant growth promotion, and isoprenoid biosynthetic machinery.

Globally, due to widespread dispersion, intraspecific diversity, and crucial ecological components of halophilic ecosystems, halophilic bacteria is considered one of the key models for ecological, adaptative, and biotechnological applications research in saline environments. With this aim, the present study was to enlighten the plant growth-promoting features and investigate the systematic genome of a halophilic bacteria, Virgibacillus halodenitrificans ASH15, through single-molecule real-time (SMRT) sequencing technology. Results showed that strain ASH15 could survive in high salinity up to 25% (w/v) NaCl concentration and express plant growth-promoting traits such as nitrogen fixation, plant growth hormones, and hydrolytic enzymes, which sustain salt stress. The results of pot experiment revealed that strain ASH15 significantly enhanced sugarcane plant growth (root shoot length and weight) under salt stress conditions. Moreover, the sequencing analysis of the strain ASH15 genome exhibited that this strain contained a circular chromosome of 3,832,903 bp with an average G+C content of 37.54%: 3721 predicted protein-coding sequences (CDSs), 24 rRNA genes, and 62 tRNA genes. Genome analysis revealed that the genes related to the synthesis and transport of compatible solutes (glycine, betaine, ectoine, hydroxyectoine, and glutamate) confirm salt stress as well as heavy metal resistance. Furthermore, functional annotation showed that the strain ASH15 encodes genes for root colonization, biofilm formation, phytohormone IAA production, nitrogen fixation, phosphate metabolism, and siderophore production, which are beneficial for plant growth promotion. Strain ASH15 also has a gene resistance to antibiotics and pathogens. In addition, analysis also revealed that the genome strain ASH15 has insertion sequences and CRISPRs, which suggest its ability to acquire new genes through horizontal gene transfer and acquire immunity to the attack of viruses. This work provides knowledge of the mechanism through which V. halodenitrificans ASH15 tolerates salt stress. Deep genome analysis, identified MVA pathway involved in biosynthesis of isoprenoids, more precisely "Squalene." Squalene has various applications, such as an antioxidant, anti-cancer agent, anti-aging agent, hemopreventive agent, anti-bacterial agent, adjuvant for vaccines and drug carriers, and detoxifier. Our findings indicated that strain ASH15 has enormous potential in industries such as in agriculture, pharmaceuticals, cosmetics, and food.

Switching carbon metabolic flux for enhancing the production of sesquiterpene-based high-density biofuel precursor in Saccharomyces cerevisiae

BackgroundSesquiterpenes are designated as a large class of plant-derived natural active compounds, which have wide applications in industries of energy, food, cosmetics, medicine and agriculture. Neither plant extraction nor chemical synthesis can meet the massive market demands and sustainable development goals. Biosynthesis in microbial cell factories represents an eco-friendly and high-efficient way. Among several microorganisms, Saccharomyces cerevisiae exhibited the potential as a chassis for bioproduction of various sesquiterpenes due to its native mevalonate pathway. However, its inefficient nature limits biosynthesis of diverse sesquiterpenes at industrial grade.ResultsHerein, we exploited an artificial synthetic malonic acid-acetoacetyl-CoA (MAAC) metabolic pathway to switch central carbon metabolic flux for stable and efficient biosynthesis of sesquiterpene-based high-density biofuel precursor in S. cerevisiae. Through investigations at transcription and metabolism levels, we revealed that strains with rewired central metabolism can devote more sugars to β-caryophyllene production. By optimizing the MVA pathway, the yield of β-caryophyllene from YQ-4 was 25.8 mg/L, which was 3 times higher than that of the initial strain YQ-1. Strain YQ-7 was obtained by introducing malonic acid metabolic pathway. Combing the optimized flask fermentation process, the target production boosted by about 13-fold, to 328 mg/L compared to that in the strain YQ-4 without malonic acid metabolic pathway.ConclusionThis designed MAAC pathway for sesquiterpene-based high-density biofuel precursor synthesis can provide an impressive cornerstone for achieving a sustainable production of renewable fuels.

Construction and Optimization of Nonclassical Isoprenoid Biosynthetic Pathways in Yeast Peroxisomes for (+)-Valencene Production.

Isoprenoids are a kind of natural product with various activities, but their plant extraction suffers low concentration. The rapid development of synthetic biology offers a sustainable route for supply of high-value-added natural products by engineering microorganisms. However, the complexity of cellular metabolism makes engineering endogenous isoprenoid biosynthetic pathways with metabolic interaction difficult. Here, for the first time, we constructed and optimized three types of isoprenoid pathways (the Haloarchaea-type, Thermoplasma-type, and isoprenoid alcohol pathway) in yeast peroxisomes for the synthesis of sesquiterpene (+)-valencene. In yeast, the Haloarchaea-type MVA pathway is more effective than the classical MVA pathway. MVK and IPK were determined to be the rate-limiting steps of the Haloarchaea-type MVA pathway, and the production of 869 mg/L (+)-valencene under fed-batch fermentation in shake flasks was realized. This work expands isoprenoid synthesis in eukaryotes and provides a more efficient pathway for isoprenoid synthesis.

Functional characterization of triterpene synthases in Cibotium barometz

Cibotium barometz (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. C. barometz produces a range of bioactive triterpenes and their metabolites. However, the biosynthetic pathway of triterpenes in C. barometz remains unknown. To clarify the origin of diverse triterpenes in C. barometz, we conducted de novo transcriptome sequencing and analysis of C. barometz rhizomes and leaves to identify the candidate genes involved in C. barometz triterpene biosynthesis. Three C. barometz triterpene synthases (CbTSs) candidate genes were obtained. All of them were highly expressed in C. barometz rhizomes, consisting of the accumulation pattern of triterpenes in C. barometz. To characterize the function of these CbTSs, we constructed a squalene- and oxidosqualene-overproducing yeast chassis by overexpressing all the enzymes in the MVA pathway under the control of GAL-regulated promoter and disrupted the GAL80 gene in Saccharomyces cerevisiae simultaneously. Heterologous expressing CbTS1, CbTS2, and CbTS3 in engineering yeast strain produced cycloartenol, dammaradiene, and diploptene, respectively. Phylogenetic analysis revealed that CbTS1 belongs to oxidosqualene cyclase, while CbTS2 and CbTS3 belong to squalene cyclase. These results decipher enzymatic mechanisms underlying the origin of diverse triterpene in C. barometz.

Hepatic Anti-Oxidative Genes CAT and GPX4 Are Epigenetically Modulated by RORγ/NRF2 in Alphacoronavirus-Exposed Piglets.

As a member of alpha-coronaviruses, PEDV could lead to severe diarrhea and dehydration in newborn piglets. Given that lipid peroxides in the liver are key mediators of cell proliferation and death, the role and regulation of endogenous lipid peroxide metabolism in response to coronavirus infection need to be illuminated. The enzymatic activities of SOD, CAT, mitochondrial complex-I, complex-III, and complex-V, along with the glutathione and ATP contents, were significantly decreased in the liver of PEDV piglets. In contrast, the lipid peroxidation biomarkers, malondialdehyde, and ROS were markedly elevated. Moreover, we found that the peroxisome metabolism was inhibited by the PEDV infection using transcriptome analysis. These down-regulated anti-oxidative genes, including GPX4, CAT, SOD1, SOD2, GCLC, and SLC7A11, were further validated by qRT-PCR and immunoblotting. Because the nuclear receptor RORγ-driven MVA pathway is critical for LPO, we provided new evidence that RORγ also controlled the genes CAT and GPX4 involved in peroxisome metabolism in the PEDV piglets. We found that RORγ directly binds to these two genes using ChIP-seq and ChIP-qPCR analysis, where PEDV strongly repressed the binding enrichments. The occupancies of histone active marks such as H3K9/27ac and H3K4me1/2, together with active co-factor p300 and polymerase II at the locus of CAT and GPX4, were significantly decreased. Importantly, PEDV infection disrupted the physical association between RORγ and NRF2, facilitating the down-regulation of the CAT and GPX4 genes at the transcriptional levels. RORγ is a potential factor in modulating the CAT and GPX4 gene expressions in the liver of PEDV piglets by interacting with NRF2 and histone modifications.

Cloning, Bioinformatics Analysis and Expression Vector Construction of <I>DXS</I> Gene from <i>Aconitum vilmorinianum</i>

<i>Aconitum vilmorinianum</i> is an important Chinese medicinal herb, which contains diterpenoid alkaloids and has the functions of antirheumatic activity, improvement in human meridians, relief of swelling and pain, activation of the blood circulation, and inhibition of tumor growth. The biosynthesis of diterpenoid alkaloids requires intermediate products obtained through terpenoid skeleton metabolism, in order to synthesize diterpenoid alkaloids. The synthesis of terpenoid skeletons includes the MVA pathway and the MEP pathway. 1-deoxyglucose-5-phosphate synthase (<I>DXS</I>) is the first rate limiting enzyme gene in the MEP pathway and plays an important regulatory role in the synthesis of terpenoids. Objective: To understand the characteristics of the DXS gene in Polygonummultiflorum. Method: By extracting RNA from the leaves of <i>A. vilmorinianum</i>and reverse transcribing it into cDNA, according to the transcriptome data of <i>A. vilmorinianum</i>, primers were designed to clone the full length of the gene, and the sequence of the coding protein was analyzed by bioinformatics. Construct overexpression vectors and inhibitory expression vectors. Results: The results showed that the coding region of the gene is 1512 bp, with 503 amino acids, molecular formula C₂₄₀₈H₃₈₂₂N₆₅₂O₇₁₃S₂₂, molecular weight 54020.10, the isoelectric point pI 6.29, the instability index 35.70, and belongs to the stable protein category. Successfully constructing overexpression vectors and inhibitory expression vectors can provide scientific basis for further research on the function of this gene in the diterpene biosynthesis pathway.

Enhancement of β-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of β-Caryophyllene Synthase and Engineering the Chassis.

β-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. β-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of β-caryophyllene synthase (CPS) is one of the main restrictive factors for β-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the β-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in Vmax and Kcat. The Kcat/Km of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in β-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to β-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6T1025N to improve the transmembrane transport of β-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of β-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a β-caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of β-caryophyllene production by yeast.

Alpha-Tocopherol Significantly Improved Squalene Production Yield of Aurantiochytrium sp. TWZ-97 through Lowering ROS levels and Up-Regulating Key Genes of Central Carbon Metabolism Pathways.

Media supplementation has proven to be an effective technique for improving byproduct yield during microbial fermentation. This study explored the impact of different concentrations of bioactive compounds, namely alpha-tocopherol, mannitol, melatonin, sesamol, ascorbic acid, and biotin, on the Aurantiochytrium sp. TWZ-97 culture. Our investigation revealed that alpha-tocopherol was the most effective compound in reducing the reactive oxygen species (ROS) burden, both directly and indirectly. Adding 0.7 g/L of alpha-tocopherol led to an 18% improvement in biomass, from 6.29 g/L to 7.42 g/L. Moreover, the squalene concentration increased from 129.8 mg/L to 240.2 mg/L, indicating an 85% improvement, while the squalene yield increased by 63.2%, from 19.82 mg/g to 32.4 mg/g. Additionally, our comparative transcriptomics analysis suggested that several genes involved in glycolysis, pentose phosphate pathway, TCA cycle, and MVA pathway were overexpressed following alpha-tocopherol supplementation. The alpha-tocopherol supplementation also lowered ROS levels by binding directly to ROS generated in the fermentation medium and indirectly by stimulating genes that encode antioxidative enzymes, thereby decreasing the ROS burden. Our findings suggest that alpha-tocopherol supplementation can be an effective method for improving squalene production in Aurantiochytrium sp. TWZ-97 culture.