129 results about "Mevalonic acid" patented technology

The invention belongs to the technical application field of microbial metabolic engineering and synthetic biology, and particularly relates to a pichia pastoris engineering strain for high-yield sclareol as well as a construction method and application of the pichia pastoris engineering strain. The method comprises the following steps: constructing a biosynthetic pathway of sclareol in a host strain, and optimizing an intracellular mevalonic acid metabolic pathway and a central metabolic pathway to obtain an engineering strain A; the host strain is pichia pastoris; or overexpressing or knocking out a metabolism regulation factor in the obtained engineering bacterium A to obtain an engineering bacterium strain B; or, in the obtained engineering bacterium B, a synthetic pathway is targeted to peroxisome and optimized by adopting a cell region compartment strategy, and an engineering bacterium strain C is obtained. The invention provides a pichia pastoris chassis cell for synthesizing a diterpenoid sclareol and an engineering strain for synthesizing sclareol. By introducing and optimizing the expression of a sclareol synthesis pathway, the fermentation yields of sclareol in shake flask batch and bioreactor batch fed-batch fermentation are respectively 631.6 mg / L and 10.5 g / L.

The present invention relates to a recombinant yeast with high squalene production, a construction method and a use thereof, wherein the recombinant yeast is a saccharomyces cerevisiae that expresses or overexpresses HMGR gene, ADA gene, LEU2 gene, MVA pathway gene and squalene synthase gene ERG9. The present invention integrates acetaldehyde dehydrogenase gene ADA and HMG-CoA reductase gene tHMGR in multiple copies in saccharomyces cerevisiae, integrates the screened high-activity phosphoketolase gene PK and phosphotransacetylase gene PTA, weakens epoxysqualene synthesis, strengthens the mevalonate pathway, overexpresses yeast endogenous squalene synthase gene, etc. to obtain a recombinant yeast with high squalene production, so that the production in the shake flask stage of the production process exceeds 5g / L, and the production in the fed batch fermentation stage exceeds 50g / L, breaking through the current bottleneck of microbial synthesis of squalene production, and is suitable for practical promotion and application.

The invention discloses a recombinant strain for producing squalene as well as a construction method and application of the recombinant strain. The construction method comprises the following steps: heterologously expressing cDNA (complementary deoxyribonucleic acid) of each gene in a squalene synthesis route from filamentous fungi in yeast; the squalene synthetic pathway genes comprise a mevalonic acid-5-phosphate kinase gene, a squalene synthase gene, an acetoacetyl coenzyme A thiolase gene, a mevalonic acid kinase gene, a hydroxymethyl glutaryl coenzyme a synthetase gene, a mevalonic acid pyrophosphate decarboxylase gene, a farnesyl pyrophosphate synthetase gene and isopentenyl pyrophosphate. A dimethallylpyrophosphate isomerase gene and a truncated hydroxymethyl glutaryl coenzyme a reductase gene; the culture medium is characterized in that the filamentous fungi are Saroocladium oryzae or Stibella sp. 1056, and the culture medium is a culture medium. The excavated squalene synthesis route can complete efficient de novo synthesis of squalene, and the squalene yield is increased.

The invention relates to recombinant saccharomyces cerevisiae for de novo synthesis of mevalonic acid as well as a construction method and application of the recombinant saccharomyces cerevisiae, and belongs to the technical field of genetically engineered bacteria. The technical problem to be solved by the invention is to provide the recombinant saccharomyces cerevisiae for de novo synthesis of mevalonic acid. According to the recombinant saccharomyces cerevisiae, saccharomyces cerevisiae is used as a chassis strain, and ACL, CTP1, RtME and MDH3 are over-expressed. A series of recombinant saccharomyces cerevisiae strains with high yield of mevalonic acid are constructed through a gene recombination method, the strains can be fermented in a conventional culture medium to prepare mevalonic acid, the fermentation process is simple, the cost is low, the yield is high, and the maximum yield of mevalonic acid can reach 5692.45 mg / L. According to the invention, the recombinant saccharomyces cerevisiae strain is used as a chassis strain, and recombinant genetically engineered bacteria for producing the santalene, the santalol and the patchouli alcohol are respectively obtained through gene recombination, so that the yields of the santalene, the santalol and the patchouli alcohol produced by fermentation are improved.

The invention discloses a high-yield delta-tocotrienol genetically engineered bacterium and application thereof, and belongs to the technical field of gene recombination. The invention discloses a genetically engineered bacterium for high yield of delta-tocotrienols, which is characterized in that yarrowia lipolytica ATCC MYA-2613 is used as an original strain, and a ku70 gene is knocked out; the gene is used for expressing and coding 4-hydroxyphenylpyruvate dioxygenase, the gene is used for coding uromelanic acid phytate transferase and the gene is used for coding tocopherol cyclase; intensifying a shikimic acid pathway; a mevalonic acid pathway is strengthened; an isopentenol path is introduced; carrying out protein fusion on the HPD and the HPT; truncating a signal peptide of a gene encoding a tocopherol cyclase and back-filling a leucine defect. The genetically engineered bacterium constructed by the invention has industrial stability, can be amplified by more than 10 times in a 2L fermentation tank level compared with a shake flask level, can produce 616.45 mg / L of delta-tocotrienol after being cultured for 240 hours, and has industrial application prospects.

The invention belongs to the technical field of biosynthesis, and particularly relates to an engineering bacterium for preparing stevioside through de novo fermentation as well as a preparation method and application of the engineering bacterium. According to the invention, mevalonate kinase in the MVA pathway is mutated and optimized. The method comprises the following steps: carrying out point mutation on mevalonate kinase MvK of a wild type source, constructing a stevioside synthesis route in an escherichia coli host, and comparing the influence of MvK mutation on the yield of stevioside, so as to determine a Q160L mutant, and applying the Q160L mutant to the stevioside production route to realize efficient production of stevioside.

The invention provides a method for biosynthesizing menadione by using yarrowia lipolytica. The method is realized by constructing recombinant yarrowia lipolytica, and the construction method of the recombinant yarrowia lipolytica for synthesizing menadione comprises the following steps: intensively expressing a mevalonic acid pathway in the yarrowia lipolytica, and synthesizing menadione by introducing a synthesis pathway of heterologous geranyl and geranyl pyrophosphate. A sufficient isoprene side chain donor is provided for biosynthesis of menadione; on the basis, an aromatic isopentenyl transferase coding gene which is optimized by codons and has menadione substrate specificity is over-expressed in yarrowia lipolytica. The construction method of the yarrowia lipolytica for biosynthesis of menadione provided by the invention is simple to operate, the constructed yarrowia lipolytica can obtain a menadione product with relatively high concentration by taking commercially available menadione as a substrate, the biosynthesis steps are relatively short, and the yarrowia lipolytica has relatively high production and application values.

The invention belongs to the technical field of genetic engineering and fermentation engineering, discloses a yarrowia lipolytica genetically engineered bacterium capable of realizing high yield of retinol and application of the yarrowia lipolytica genetically engineered bacterium, and particularly discloses a yarrowia lipolytica genetically engineered bacterium capable of realizing high yield of retinol. In the engineering strain, hyphae gene and lipid peroxidation gene knockout is carried out to construct a retinol synthesis pathway, and / or strengthen a yarrowia lipolytica endogenous mevalonic acid pathway, and / or strengthen key gene expression of a yarrowia lipolytica exogenous carotenoid synthesis pathway at the same time, so that high yield of retinol can be effectively realized.

The invention belongs to the technical field of biology, and particularly relates to recombinant escherichia coli for producing melonic acid and application of the recombinant escherichia coli. Escherichia coli MG1655 is used as a host cell, and HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene derived from lactobacillus casei, acetyl-coenzyme A acetyltransferase atoB gene derived from escherichia coli, phosphoketoenzyme fxpk gene derived from bifidobacterium adolescentis and fructose-1, 3-D-glucanase derived from escherichia coli are synthesized into an HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene derived from lactobacillus casei. And transferring the 2, 6-diphosphatase fbp gene into Escherichia coli host cells to obtain the gene. The recombinant Escherichia coli constructed by the invention can be used for synthesis of the melonic acid, the yield of the melonic acid can reach 87.86 g / L after the recombinant Escherichia coli takes glucose as a substrate and is fermented for 48 hours in a 5L fermentation tank at 37 DEG C, and the recombinant Escherichia coli has the advantages of short fermentation time and high yield and meets the requirements of industrial production.

The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae engineering strain for producing caryophyllene and application of the saccharomyces cerevisiae engineering strain. The invention provides a saccharomyces cerevisiae engineering strain which has enhanced expression and / or activity of mevalonic acid pathway enzyme and one or more modifications selected from the following (1)-(2): (1) weakened expression and / or activity of sterol synthesis pathway enzyme; (2) attenuated expression and / or activity of fatty acid synthetic pathway enzymes; compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantage that the sesquiterpene yield is increased. Compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantages that the production capacity of the sesquiterpenes such as the caryophyllene is obviously improved, the sesquiterpenes such as the caryophyllene can be efficiently synthesized, and the saccharomyces cerevisiae engineering strain has better application value.

The invention belongs to the technical field of bioengineering, and relates to a construction method and application of recombinant yarrowia lipolytica for synthesizing plant antitoxin Kauralexin A1. The method is realized by constructing recombinant yarrowia lipolytica. The construction method of the recombinant yarrowia lipolytica for synthesizing the Kauralexin A1 comprises the following steps: overexpressing a codon optimized antitoxin Kauralexin A1 biosynthetic pathway in yarrowia lipolytica, screening adaptive P450 reductase to improve the electron transfer efficiency in a P450 system, and constructing a metabolic pathway scaffold-free multi-enzyme complex to enhance cascade biological catalysis and metabolic flux, so as to obtain the recombinant yarrowia lipolytica for synthesizing the Kauralexin A1. The expression of the mevalonic acid pathway is enhanced, so that a sufficient GGPP precursor is provided for the biosynthesis of the Kauralexin A1. The construction method of the yarrowia lipolytica for biosynthesizing the antitoxin Kauralexin A1, provided by the invention, is simple to operate, and the constructed yarrowia lipolytica can be used for efficiently producing the Kauralexin A1 and has relatively high production and application values.

The present invention relates to the field of bioengineering, and in particular to an engineering bacterium with high yield of astaxanthin and its construction method and application. The present invention starts with wild-type Yarrowia lipolytica, introduces four genes of carB, carRP, crtZ and crBKT through a non-homologous recombination strategy, further overexpresses the key enzyme encoding gene in the mevalonate pathway (MVA) pathway to increase the yield of astaxanthin, and realizes the efficient production of the natural product astaxanthin. At the same time, the recombinant Yarrowia lipolytica provided by the present invention uses copper ion induction expression promoter MT-2 to control the expression of β-carotene ketolase crBKT gene, so that it keeps spatiotemporal differential expression with β-carotene hydroxylase gene crtZ, and when high concentration of intracellular zeaxanthin accumulates, copper ions are added to induce the expression of β-carotene ketolase crBKT gene, reduce the production of intermediate metabolic byproducts, and promote the efficient production of astaxanthin.

The present invention belongs to the field of microbial metabolic engineering and synthetic biology technology applications, and particularly relates to a Pichia pastoris engineered strain that produces high sclareol production, as well as its construction method and application. A sclareol biosynthesis pathway is constructed in a host strain, and the intracellular mevalonate metabolic pathway and the central metabolic pathway are optimized, thereby obtaining an engineered bacterial strain A; the host strain is Pichia pastoris; or, metabolic regulatory factors are overexpressed or knocked out in the above-obtained engineered bacterial strain A, thereby obtaining an engineered bacterial strain B; or, in the above-obtained engineered bacterial strain B, a cell compartmentalization strategy is used to target the synthesis pathway to the peroxisome and optimize it, thereby obtaining an engineered bacterial strain C. The present invention provides a Pichia pastoris chassis cell for synthesizing the diterpenoid compound sclareol and an engineered strain for synthesizing sclareol; by introducing and optimizing the expression of the sclareol synthesis pathway, the sclareol yields in shake flask batch fermentation and bioreactor batch fed-batch fermentation reach 631.6 mg / L and 10.5 g / L, respectively.

The invention discloses a recombinant bacterium capable of increasing the yield of alpha-bisabolol as well as a preparation method and application of the recombinant bacterium, and belongs to the technical field of microorganisms. The invention aims to improve the yield of alpha-bisabolol and enhance the tolerance of a host to an organic solvent. The invention provides a recombinant bacterium for improving the yield of alpha-bisabolol. Escherichia coli is used as a starting strain; the method comprises the following steps of: overexpressing an acetyl CoA acyltransferase / HMG-CoA reductase mvaE gene, an HMG-CoA synthetase mvaS gene, a 2-methyl citrate dehydratase prpD gene, a mevalonate kinase ERG12 gene, a mevalonate 5-phosphate kinase ERG8 gene, a mevalonate 5-diphosphate decarboxylase ERG19 gene and an isopentenyl diphosphate isomerase idi gene, so as to obtain a recombinant vector; the gene is obtained from an alpha-bisabolol synthase gene of artichoke, a farnesyl diphosphate synthase ispA gene and an alpha-bisabolol synthase CcBOS gene of artichoke. The industrial process of synthesizing alpha-bisabolol by a biological method is promoted.

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for de novo biosynthesis of cycloastragenol as well as a construction method and application of the engineering bacterium. A mevalonic acid (MVA) pathway is reconstructed in a saccharomyces cerevisiae strain peroxidase body by utilizing a cell region compartment strategy, a key enzyme gene of the MVA pathway is over-expressed by using a strong promoter, and the cycloastragenol is obtained. The key enzyme genes are used as expression elements of cycloastragenol, are introduced into the chassis saccharomyces cerevisiae through a CRISPR-Cas9 technology, and are integrated on a saccharomyces cerevisiae genome through homologous recombination, so that a saccharomyces cerevisiae engineering bacterium CycZ23 and an engineering bacterium CAG-1 for efficiently synthesizing cycloastragenol are constructed, and de novo synthesis of plant triterpenoid natural products is realized; the highest titer of the generated cycloastragenol is 236 mg / L; the synthesis method is economical, efficient, green, environmentally friendly and easy to operate, and has important application value.

The invention discloses an NK cell serum-free medium, the serum-free medium comprises a basic medium and an additive, the additive comprises the following components: recombinant human transferrin, recombinant human insulin, human serum albumin, growth factors, glucose, trace elements, tacrolimus and mevalonic acid, the serum-free culture medium comprises the following components in percentage by weight: 85% of a basic culture medium, 3% of recombinant human transferrin, 3% of recombinant human insulin, 2% of human serum albumin, 2% of growth factors, 2% of glucose, 1.5% of trace elements, 1% of tacrolimus and 0.5% of mevalonic acid; according to the serum-free culture medium for the NK cells, growth and differentiation of the NK cells can be adjusted by adding a proper amount of tacrolimus, meanwhile, a proper amount of mevalonic acid is matched, raw materials of active substances can be provided for the cultured NK cells, growth and development of the NK cells or expression of specific functions are facilitated, and therefore the survival rate and the amplification number of the NK cells can be increased.

The invention discloses a key enzyme farnesyl pyrophosphate synthase of a black fungus terpenoid synthetic route, and belongs to the technical field of protein. The invention aims to provide a key enzyme gene of a mevalonic acid pathway synthesis pathway in black fungus. The invention provides an FPPS protein of black fungus. The amino acid sequence of the FPPS protein is as shown in SEQ ID NO. 2. A solid foundation is laid for deeply exploring functions of key genes for triterpene synthesis, and meanwhile important data support is provided for systematically explaining specific effects of the genes in terpene synthesis routes.

The invention is related to a process for synthesizing a sustainable aviation fuel composition. The process comprises providing a reaction mixture comprising a first compound that is at least one of mevalonolactone, mevalonic acid, mevalonate salt, dehydromevalonic acid, dehydromevalonate salt, dehydromevalonolactone or combinations thereof. The process then involves converting the first compound in the reaction mixture to provide a first intermediate comprising isoprene. Then, the isoprene in the first intermediate is reacted in the presence of a first heat transfer agent to provide a second intermediate comprising terpenes. Finally, the second intermediate is allowed to react in the presence of a second heat transfer agent, or alternatively in neat conditions, and optionally in presence of a catalyst to provide the sustainable fuel composition. The sustainable aviation fuel composition made available from the process of the invention is found to comprise monocyclic aromatic hydrocarbons (MAHs) at useful concentration ranges (along with cycloalkanes) while they are substantially devoid of Polycyclic aromatic hydrocarbons (PAHs).

The present invention discloses a method for promoting the synthesis of 7-dehydrocholesterol by Saccharomyces cerevisiae. By mutating phosphomevalonate kinase ERG8 and squalene synthase ERG9 and integrating them into the Saccharomyces cerevisiae genome separately or simultaneously, several mutant strains with significantly increased 7-dehydrocholesterol production are obtained. Among them, phosphomevalonate kinase ERG8 involves mutations at one or more sites such as 75, 187, 192, 205, 206, 314, 322, 349, etc., and squalene synthase ERG9 involves mutations at one or more sites such as 96, 108, 146, 208, 209, 251, 252, 258, 286, 345, 355, 388, 389, 404, 426, 434, etc. The Saccharomyces cerevisiae mutants provided by the present invention can achieve the production of 7-DHC, laying a foundation for metabolic engineering transformation of Saccharomyces cerevisiae to promote 7-DHC synthesis.

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to yarrowia lipolytica engineering bacteria for producing zeaxanthin as well as a construction method and application of the yarrowia lipolytica engineering bacteria. Specifically, the yarrowia lipolytica is subjected to genetic engineering modification based on a genetic engineering technology, a zeaxanthin biosynthetic pathway is constructed by expressing beta-carotene hydroxylase in an original strain, and meanwhile, a zeaxanthin biosynthetic pathway is constructed through a multiple metabolic engineering modification strategy. The method comprises the steps of key enzyme PaCrtZ multi-copy integration, oxidation-reduction balance regulation and control, upstream mevalonic acid pathway enhancement through membrane-free organelles and the like, and the yield of zeaxanthin is further increased. Meanwhile, by optimizing the culture conditions and applying the optimal fermentation conditions to perform tank fermentation, the yarrowia lipolytica can continuously generate more zeaxanthin, so that the yarrowia lipolytica has excellent practical application value and application prospect.

The present invention relates to host cells comprising genes of the mevalonate and Nudix pathways, engineered fusion proteins of enzymes of the mevalonate and Nudix pathways, methods as well as kits for producing geraniol and geranyl acetate.

The invention discloses a recombinant bacterium for producing mevalonic acid by taking glucose and acetone as co-substrates as well as a construction method and application of the recombinant bacterium, and belongs to the technical field of genetic engineering. The problems that an existing mevalonic acid biosynthesis mode is low in carbon atom economy, limited in theoretical yield and the like are solved. According to the invention, acetone carboxylase, acetoacetyl coenzyme A synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase and hydroxymethyl glutaryl-CoA synthetase are subjected to heterologous expression in escherichia coli with an acetyl coenzyme A acetyltransferase gene atoB and a histidine protein kinase gene atoS knocked out, and self carbonic anhydrase is over-expressed; and a new way for synthesizing MVA by using glucose and acetone as a co-substrate is constructed. According to the method, one molecule of CO2 can be fixed in the biosynthesis of mevalonic acid, the yield of carbon atoms in the synthesis process of mevalonic acid is increased, the problem of low yield of carbon atoms in the synthesis process of mevalonic acid by taking glucose as a single substrate is solved, and a new thought is provided for constructing a carbon neutralization type biological manufacturing technology.

The present invention provides a recombinant microorganism for simultaneously producing sesquiterpenes and primary metabolites, wherein the primary metabolites include at least one of pantothenic acid, squalene, fatty acids and vitamin B2; the strain contains the mevalonate pathway, the β-farnesene synthesis gene and genes related to the synthesis of primary metabolites; the genes related to the mevalonate pathway include ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1 and ERG20 genes, and the copy numbers are ERG10:ERG13:tHMG1:ERG12:ERG8:MVD1:IDI1:ERG20 = 2:2:X:2:2:2:2:2, where X is an integer greater than or equal to 1; the β-farnesene synthesis gene includes bFS, and the sequence is as shown in SEQ ID NO.1. The present invention realizes high yield of primary metabolites while producing sesquiterpene compounds.

The present application relates to the promotion oil bean root secretion of mevalonic acid microbial source humic acid fertilizer, the promotion oil bean root secretion of mevalonic acid artificial synthesis microbial source humic acid is prepared by the following method: the corn stalk is predecomposed by using the big ball cover mushroom strain, and the corn slurry with the concentration of 0-1% is added, the moisture content of corn stalk is 55-65%, the predecomposed corn stalk is added with water, is handled with water treatment device, and the mixture containing microbial fungal residues is obtained;The mixture containing microbial fungal residues is converted into artificial synthesis microbial source humic acid by four-stage reaction;The artificial synthesis microbial source humic acid fertilizer is applied to the root of oil bean, the mevalonic acid secreted by the root of oil bean reaches 0.006-0.007 ng / mg, and the length of the root of oil bean reaches 25-26 cm.The present application can improve the content of mevalonic acid secreted by the root of oil bean, improve the yield and quality of oil bean.

The present invention provides a use of mevalonic acid in the manufacture of a drug for improving early embryonic development of aged oocytes, which belongs to the field of biomedicine technology. Through experimental research, the present invention has found that mevalonic acid can be used to improve the fertilization rate, two-cell formation rate and good-quality blastocyst formation rate of aged oocytes, thereby improving the development process of early embryos, providing a new idea and method for infertility treatment.

The invention belongs to the technical field of enzyme engineering, and discloses a high-activity isopentenyl phosphokinase mutant and application thereof. The high-activity isopentenyl phosphokinase mutant is AtIPKH274P, and the amino acid at the 274 site of wild type AtIPK is mutated from histidine H to proline P. The invention further discloses a preparation method of the high-activity isopentenyl phosphokinase mutant. According to the present invention, the AtIPKH274P, the SmDAGKS47A and the L124A can form the efficient isopentenol utilization pathway so as to effectively convert the prenol into the DMAPP; an engineering strain with improved isopentenyl modification capability can be constructed by utilizing the approach, and the strain has more advantages compared with a traditional engineering strain based on a mevalonic acid approach.

The invention discloses a multi-gene tandem recombinant strain as well as a construction method and application thereof. The invention also discloses a method for synthesizing lycopene based on an isopentenol utilization approach. The strain comprises a recombinant strain of seven key genes, namely mtipk, thim, ispA, idi, crtE, crttheB and crtI, for synthesizing lycopene in an IUP (Inhibitory Urinary Protein) way. According to the invention, the isopentenol is introduced into the escherichia coli in a way to synthesize the lycopene; according to the approach, only two-step phosphorylation reaction is used for replacing the original 2-C-methyl-D-erythritol-4-phosphoric acid approach and mevalonic acid approach which need more than seven steps to synthesize precusor substances pentadienyl pyrophosphoric acid and dimethyl allyl pyrophosphate ester of lycopene, so that the metabolic pathway is greatly shortened, and competition with central carbon metabolism in cells is avoided; the toxicity caused by accumulation of intermediate products in the metabolic process is reduced, and lycopene can be efficiently synthesized by using a cheap substrate.

The utility model relates to the technical field of preparation and cultivation of melonic acid, in particular to a fermentation tank convenient to adjust and use for preparation and cultivation of melonic acid, which comprises a fermentation tank body, a cover plate arranged at the upper end of the fermentation tank body, a first feed pipe arranged on one side of the cover plate, a motor mounted on the upper side of the cover plate, and a rotating pipe arranged at the output end of the motor. The outer side of the rotating pipe is sleeved with a first annular piece, a plurality of first arc-shaped plates are arranged on the outer side of the first annular piece, a plurality of nozzles are formed in the first arc-shaped plates, and a PH detector is installed on the fermentation tank body. According to the utility model, a solution in the fermentation tank body can be detected in time through the PH detector, then ammonia water enters the cylinder, the ammonia water is discharged to the middle of the solution in the fermentation tank body through the plurality of nozzles, and the ammonia water and the solution are rapidly mixed through the rotation of the plurality of first arc-shaped plates, so that the PH value of the solution is rapidly adjusted.

This invention belongs to the field of microbial metabolic engineering and biotechnology applications, specifically relating to an engineered bacterium for synthesizing monoterpenoid compounds, its construction method, and its applications. The construction method involves using *Hansenula polymorpha* as a host, regulating the mevalonate pathway in its cytoplasm, and overexpressing monoterpene synthase to obtain the engineered bacterium; or, overexpressing monoterpene synthase and the mevalonate pathway in peroxisomes to obtain the engineered bacterium. The engineered bacterium constructed in this application can efficiently synthesize monoterpenoid compounds such as geraniol, β-myrcene, and dextrorotatory borneol.

The invention provides application of a group of plasma metabolism markers in preparation of a reagent product for pulmonary nodule diagnosis, and relates to the technical field of disease diagnosis reagents. The metabolic marker is prepared from trigonelline, 4-methylcatechol, 2, 4-dinitrophenol, 3, 4-dihydroxyphenylacetic acid, N-(2-furfuryl) glycine, mevalonic acid and formononetin, and the metabolic marker is prepared from the following raw materials: the trigonelline, the 4-methylcatechol, the 2, 4-dinitrophenol, the 3, 4-dihydroxyphenylacetic acid, the N-( According to the application, ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC / MS) is adopted to carry out metabonomics analysis on plasma samples before and after an early pulmonary nodule operation, and a group of plasma metabolites (trigonelline, 4-methylcatechol, 2, 4-dinitrophenol, 3, 4-dihydroxyphenylacetic acid, N-(2-furfuryl) glycine, mevalonic acid and formononetin) are identified; and the group of metabolites is utilized to form a diagnosis model, so that benign and malignant pulmonary nodules can be accurately diagnosed, and early-stage differential diagnosis and risk assessment are facilitated.