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1.
Front Genet ; 13: 894928, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35547255

RESUMO

MYB superfamily is one of the most abundant families in plants, and plays critical role in plant growth, development, metabolism regulation, and stress response. Curcuma wenyujin is the main source plant of three traditional Chinese medicines, which are widely used in clinical treatment due to its diverse pharmacological activities. In present study, 88 CwMYBs were identified and analyzed in C. wenyujin, including 43 MYB-related genes, 42 R2R3-MYB genes, two 3R-MYB genes, and one 4R-MYB gene. Forty-three MYB-related proteins were classified into several types based on conserved domains and specific motifs, including CCA1-like type, R-R type, Myb-CC type, GARP-like type, and TBR-like type. The analysis of motifs in MYB DBD and no-MYB regions revealed the relevance of protein structure and function. Comparative phylogeny analysis divided 42 R2R3-MYB proteins into 19 subgroups and provided a reference for understanding the functions of some CwMYBs based on orthologs of previously characterized MYBs. Expression profile analysis of CwMYB genes revealed the differentially expressed genes responding to various abiotic stresses. Four candidate MYB genes were identified by combining the results of phylogeny analysis and expression analysis. CwMYB10, CwMYB18, CwMYB39, and CwMYB41 were significantly induced by cold, NaCl, and MeJA stress treatments. CwMYB18 and CwMYB41 were proved as regulators with activity of transcriptional activation, whereas CwMYB39 and CwMYB10 were not. They may participate in the response to abiotic stresses through different mechanisms in C. wenyujin. This study was the first step toward understanding the CwMYB family and the response to abiotic stresses in C. wenyujin.

2.
Synth Syst Biotechnol ; 7(3): 849-861, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35572764

RESUMO

Leaf and tuber extracts of Curcuma wenyujin contain a mixture of curcuminoids. However, the curcuminoid constituents and their molecular mechanisms are poorly understood, and the relevant curcumin synthases remain unclear. In this study, we comprehensively compared the metabolite profiles of the leaf and tuber tissues of C. wenyujin. A total of 11 curcuminoid metabolites were identified and exhibited differentially changed contents in the leaf and tuber tissues. An integrated analysis of metabolomic and transcriptomic data revealed the proposed biosynthesis pathway of curcuminoid. Two candidate type Ⅲ polyketide synthases (PKSs) were identified in the metabolically engineering yeasts, indicating that CwPKS1 and CwPKS2 maintained substrate and product specificities. Especially, CwPKS1 is the first type Ⅲ PKS identified to synthesize hydrogenated derivatives of curcuminoid, dihydrocurcumin and tetrehydrocurcumin. Interestingly, the substitution of the glycine at position 219 with aspartic acid (G219D mutant) resulted in the complete inactivation of CwPKS1. Our results provide the first comparative metabolome analysis of C. wenyujin and functionally identified type Ⅲ PKSs, giving valuable information for curcuminoids biosynthesis.

4.
Cell Stem Cell ; 29(3): 386-399.e7, 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35108519

RESUMO

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth in vitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis in vivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.


Assuntos
Leucemia Mieloide Aguda , Selênio , Animais , Carcinogênese/genética , Elementos Facilitadores Genéticos/genética , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Oncogenes , Selênio/uso terapêutico
5.
Front Bioeng Biotechnol ; 10: 805429, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35198543

RESUMO

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.

6.
Plant J ; 109(3): 555-567, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34750899

RESUMO

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.


Assuntos
Celastrus/genética , Celastrus/metabolismo , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Triterpenos Pentacíclicos/metabolismo , Tripterygium/genética , Tripterygium/metabolismo , Vias Biossintéticas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Plantas Medicinais/genética , Plantas Medicinais/metabolismo
7.
Int J Biol Macromol ; 185: 949-958, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34237366

RESUMO

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Tripterygium/enzimologia , Alquil e Aril Transferases/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Terpenos/metabolismo , Tripterygium/genética
8.
Stem Cell Reports ; 16(8): 2014-2028, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34242617

RESUMO

Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Células Eritroides/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Chaperonas de Histonas/genética , Fatores de Transcrição/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Autorrenovação Celular/genética , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Hematopoese/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA-Seq/métodos , Fatores de Transcrição/metabolismo
9.
Phytochemistry ; 190: 112868, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34273756

RESUMO

Celastrol, a potent anticancer and anti-obesity drug, was first isolated from Tripterygium wilfordii Hook. f. and it is produced in small quantities in many members of the Celastraceae family. The heterologous reconstitution of celastrol biosynthesis could be a promising method for the efficient production of celastrol and natural and unnatural derivatives thereof, yet only part of the biosynthetic pathway is known. Here, we report a cytochrome P450 monooxygenase (TwCYP712K1) from T. wilfordii that performs the three-step oxidation of friedelin to polpunonic acid in the celastrol pathway. Heterologous expression of TwCYP712K1 showed that TwCYP712K1 catalyses not only the transformation of friedelin to polpunonic acid but also the oxidation of ß-amyrin or α-amyrin. The role of TwCYP712K1 in the biosynthesis of celastrol was further revealed via RNA interference. Some key residues of TwCYP712K1 were also screened by molecular docking and site-directed mutagenesis. Our results lay a solid foundation for further elucidating the biosynthesis of celastrol and related triterpenoids.


Assuntos
Triterpenos , Catálise , Sistema Enzimático do Citocromo P-450/genética , Simulação de Acoplamento Molecular , Triterpenos Pentacíclicos , Tripterygium/genética
10.
Sci Adv ; 7(30)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34290089

RESUMO

Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.


Assuntos
Leucemia Mieloide Aguda , Nicotinamida-Nucleotídeo Adenililtransferase , Animais , Apoptose/genética , Homeostase , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , NAD/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo
12.
Transl Oncol ; 14(8): 101119, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34000643

RESUMO

Relapse of childhood AML1-ETO (AE) acute myeloid leukemia is the most common cause of treatment failure. Optimized minimal residual disease monitoring methods is required to prevent relapse. In this study, we used next-generation sequencing to identify the breakpoints in the fusion gene and the DNA-based droplet digital PCR (ddPCR) method was used for dynamic monitoring of AE-DNA. The ddPCR technique provides more sensitive and precise quantitation of the AE gene during disease progression and relapse. Quantification of the AE fusion gene by ddPCR further contributes to improved prognosis. Our study provides valuable methods for dynamic surveillance of AE fusion DNA and assistance in determining the prognosis.

13.
Chemphyschem ; 22(4): 419-429, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33502098

RESUMO

Lithium-sulfur batteries with high energy density are considered as one of the most promising future energy storage devices. However, the parasitic lithium polysulfides shuttle phenomenon severely hinders the commercialization of such batteries. Ionic liquids have been found to suppress the lithium polysulfides solubility, diminishing the shuttle effect effectively. Herein, we performed classical molecular dynamics simulations to explore the microscopic mechanism and transport behaviors of typical Li2 S8 species in ionic liquids and ionic liquid-based electrolyte systems. We found that the trifluoromethanesulfonate anions ([OTf]- ) exhibit higher coordination strength with lithium ions compared with bis(trifluoromethanesulfonyl)imide anions ([TFSI]- ) in static microstructures. However, the dynamical characteristics indicate that the presence of the [OTf]- anions in ionic liquid electrolytes bring faster Li+ exchange rate and easier dissociation of Li+ solvation structures. Our simulation models offer a significant guidance to future studies on designing ionic liquid electrolytes for lithium-sulfur batteries.

14.
Plant Commun ; 2(1): 100113, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33511345

RESUMO

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.


Assuntos
Mapeamento Cromossômico , Genoma de Planta , Ginsenosídeos/biossíntese , Ginsenosídeos/genética , Panax notoginseng/genética , Panax notoginseng/metabolismo , China , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Extratos Vegetais/biossíntese , Extratos Vegetais/genética , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Transcriptoma
16.
Nat Commun ; 11(1): 5327, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087716

RESUMO

Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection. Precise characterization of clonal architecture is essential to understand the evolutionary history of tumor development and its association with treatment resistance. Here, using a single-cell DNA sequencing, we report the clonal architecture and mutational histories of 123 acute myeloid leukemia (AML) patients. The single-cell data reveals cell-level mutation co-occurrence and enables reconstruction of mutational histories characterized by linear and branching patterns of clonal evolution, with the latter including convergent evolution. Through xenotransplantion, we show leukemia initiating capabilities of individual subclones evolving in parallel. Also, by simultaneous single-cell DNA and cell surface protein analysis, we illustrate both genetic and phenotypic evolution in AML. Lastly, single-cell analysis of longitudinal samples reveals underlying evolutionary process of therapeutic resistance. Together, these data unravel clonal diversity and evolution patterns of AML, and highlight their clinical relevance in the era of precision medicine.


Assuntos
Evolução Clonal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Idoso , Animais , Evolução Clonal/efeitos dos fármacos , Estudos de Coortes , Feminino , Estudos de Associação Genética , Genômica , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Modelos Genéticos , Mutação , Análise de Sequência de DNA , Análise de Célula Única
18.
Nat Cell Biol ; 22(10): 1162-1169, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32958856

RESUMO

Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life1-3. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)-niche interaction and determines the fate of HSCs. The SEL1L-HRD1 complex, the most conserved branch of ERAD4, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity5, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell-niche interaction.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Receptores de Trombopoetina/metabolismo , Nicho de Células-Tronco , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Trombopoetina/genética , Ubiquitina-Proteína Ligases/genética
19.
Zhongguo Zhong Yao Za Zhi ; 45(13): 3098-3103, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32726017

RESUMO

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.


Assuntos
Salvia miltiorrhiza , Abietanos , Vias Biossintéticas , Raízes de Plantas
20.
Metab Eng ; 60: 87-96, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268192

RESUMO

Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.


Assuntos
Diterpenos/metabolismo , Engenharia Metabólica/métodos , Proteínas Mutantes Quiméricas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Vias Biossintéticas , Sistemas CRISPR-Cas , Simulação por Computador , Diterpenos/química , Fermentação , Redes e Vias Metabólicas , Proteínas Mutantes Quiméricas/genética , Mutação , Plasmídeos
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