产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化

瓦伦西亚烯是一种倍半萜类化合物,广泛应用于香水、香皂、食品和饮料等工业制造上。但由于其自然含量极低,且目前获取瓦伦西亚烯的方法较为麻烦且花费高,因而构建细胞工厂进行瓦伦西亚烯的生物合成是更为高效和环保的方法。选取酿酒酵母(Saccharomyces cerevisiae)作为宿主构建细胞工厂,先在酿酒酵母基因组上引入黄扁柏的瓦伦西亚烯合成酶(Valencene synthase from Callitropsis nootkatensis,CnVS),实现瓦伦西亚烯的初步合成,初始产量为4.16 mg/L。随后利用CRISPR/Cas9系统对酿酒酵母中Mevalonate(MVA)途径的erg9和rox1基因进行敲除,提高通往瓦伦西亚烯合成的碳流量。不同碳氮源浓度发酵的结果表明,细胞生长积累过高可能不利于瓦伦西亚烯的积累。最后探究了不同CnVS表达载体对瓦伦西亚烯产量的影响,并获得17.54 mg/L的最高产量,是出发菌株的4.2倍。     

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large‐scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm‐specific TPS‐d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14‐fold to 352 mg/L, bringing production to levels with industrial potential.     

Conversion of carbon dioxide into valencene and other sesquiterpenes with metabolic engineered Synechocystis sp. PCC 6803 cell factories

Valencene is a natural sesquiterpene with desirable bioactivity and aroma, making it a valuable ingredient in the food and cosmetics industries. Traditionally, valencene was extracted from the citrus fruits, and its applications were restricted by the low concentrations in natural sources and high costs for extraction. Photosynthetic biomanufacturing represents a promising route for efficient and stable production of valencene, while cyanobacteria have been considered one of the most promising platforms regarding biotechnological routes for the direct conversion of CO₂. In this work, we engineered Synechocystis sp. PCC 6803 to synthesize valencene. By introducing a heterologous valencene synthase and modifying the native MEP pathway, we obtained an efficient cyanobacterial cell factory that produced 154 mg/L valencene during a semi-continual cultivation, with an average productivity of 4.3 mg/L/day, and the cell factory exhibited robust growth and production in non-sterilized conditions. We also achieved the production of other sesquiterpenes including bisabolene, amorpha-4,11-diene, farnesene, and nerolidol by engineered cyanobacteria with enhanced MEP pathway flux, showing promising potentials as a universal chassis.     

Accumulation of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus paradisi,Citrus limonia and Citrus aurantium

The production of the sesquiterpenes nootkatone and valencene by callus cultures of Citrus species is described. The levels of these compounds were examined by gas chromatography-mass spectrometry and their yields were compared with the amounts found in mature fruits. A simultaneous increase and decrease in the levels of nootkatone and valencene, respectively, were observed with the aging of callus cultures of Citrus paradisi. These results suggest that valencene might be a possible precursor of nootkatone in this species. The high level of nootkatone detected in 9-month-old callus cultures of Citrus paradisi might be associated with the corresponding cell morphological changes observed.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FID flame-ionisation detector - FW fresh weight - GLC gas liquid chromatography - K Kinetin - NAA naphthalene acetic acid     

Harnessing yeast subcellular compartments for the production of plant terpenoids

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds.     

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144 ± 10 μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.     

香根草挥发物化学成分的分析

The chemical components of the volatiles from Vetiveria zizanioides were analyzed by SPME and GC-MS. In the roots, the main component was valencene (30.36%) abstract, while in the shoots and leaves, they were 9-octadecenamide (33.50%) abstract, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene (27.46%) abstract, and 1,2-benzendicarboxylic acid, diisooctyl ester(18.29%) abstract The results showed that there were many terpenoids in the volatils. In shoot volatiles, there existed 3 monoterpenes, 2 sesquiterpenes and 1 triterpene. Most of the volatiles in roots were sesquiterpenes.     

香根草挥发物化学成分的分析

The chemical components of the volatiles from Vetiveria zizanioides were analyzed by SPME and GC-MS. In the roots, the main component was valencene (30.36%), while in the shoots and leaves, they were 9-octadece-namide (33.50 % ), 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene (27.46% ), and 1,2-benzendicarboxylic acid, diisooctyl ester(18.29% ). The results showed that there were many terpenoids in the volatils. In shoot volatiles, there existed 3 monoterpenes, 2 sesquiterpenes and 1 triterpene. Most of the volatiles in roots were sesquiterpenes.     

Solar UV-B radiation modifies the proportion of volatile organic compounds in flowers of field-grown grapevine (Vitis vinifera L.) cv. Malbec

Ultraviolet-B solar radiation (UV-B) is an environmental signal with biological effects in different plant tissues. Recent investigations reported dramatic changes of terpenes with a protective role in plant tissues submitted to biotic and abiotic stresses. This study examined the volatile organic compounds (VOCs) profile in flowers of Vitis vinifera L. cv. Malbec under filtered UV-B (or not). Gas chromatography–electron impact mass spectrometry analysis of flowers resulted in the identification of 12 VOCs, including eight sesquiterpenes, two aldehydes, and two ketones, being the oxygenated sesquiterpene farnesol the most abundant. The total amount of VOCs in flowers did not change irrespective UV-B had been filtered or not, suggesting those compounds have a protective role that is constitutive of the reproductive tissues. However UV-B increases the proportion of valencene, β-farnesene, α-panasinsene and hepatriacontanedione which would require further investigation.     

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.     

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.     

Chemical variability of the volatile metabolites from the Caribbean corals of the genus Gorgonia

The chemical composition of the investigated gorgonians showed a high level of individual variation and the colonies, according to their major contributors, were assigned to 10 distinct chemical profiles, among which A, C, E, and G were the most abundant ones. From the metabolites identified in the present study, either by means of GC/MS or using NMR techniques after conventional separation procedures, the novel cyclic ether 5,10-epoxymuurolane is found in significant quantities in D and I chemical profiles. Furanotriene, isofuranotriene and furanodiene could be referred as the most common metabolites of the genus, since they are found in 6 out of 10 chemical profiles. Isosericenine is, also, a significant contributor of H and I chemical profiles. A number of sesquiterpene hydrocarbons, such as curzerene, bicyclogermacrene, valencene, beta-bourbonene and beta-elemene, along with the oxygenated sesquiterpenes elemanolide and furoventalene, are present at varying concentrations in the majority of the chemical profiles. Metabolites of high discriminant value are: alpha-himachalene for the K chemical profile, alpha-santalene and its oxygenated derivatives for the G chemical profile and the three geometrical isomers of germacrone for the F chemical profile. Several chemical profiles showed narrow geographic distribution. Most of the chemical profiles are located in the north, while F inhabits mainly southern sites and the others are equally distributed. Finally, 91% of the chemical profiles of the gorgonian colonies appeared to grow in all depths, while 9% did not inhabit deep-water environments. Most chemical profiles are less frequent at higher water depths with the exception of chemical profiles A and C.     

Production of the Inaccessible Sesquiterpene (−)‐5‐Epieremophilene by Metabolically Engineered Escherichia coli

(?)‐5‐Epieremophilene, an epimer of the versatile sesquiterpene (+)‐valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1‐3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (?)‐5‐epieremophilene production with yields of 42.4–76.0 mg/L in shake‐flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1‐FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (?)‐5‐epieremophilene were achieved. Biological assay indicated that (?)‐5‐epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC₅₀=1.25 μg/cm²), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (?)‐5‐epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.     

Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae

Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process.     

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208 mg L⁻¹ cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.     

陕西秦岭半仓山蝎蛉的生物学观察

2004~2005年,在陕西省南部米仓山黎坪国家森林公园研究了秦岭蝎蛉Panorpa qinlingensisChouetRan的年生活史和生物学习性,通过饲养,成功获得卵、幼虫、蛹等虫态。结果表明,秦岭蝎蛉在米仓山1年发生2代,以预蛹在土室里越冬,5月中旬始见越冬代成虫,5月中旬末~6月上旬为越冬代成虫羽化盛期,7月下旬~8月中旬为第1代成虫羽化盛期,成虫在室内可存活35~60 d。羽化近1周后开始交尾,交尾4~8 d后开始产卵,单雌产卵量35~180粒,卵期5~9 d;幼虫型,共4龄,历期38~50d;蛹为强颚离蛹,蛹期8~18 d。记述了主要生物学习性。