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Method for heterologous biosynthesis of ganoderic acid through synthetic biological means

A technology of synthetic biology and biosynthesis, applied in the field of bioengineering, can solve problems such as slow growth of the host

Active Publication Date: 2018-12-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the slow growth of the host of the existing artificially cultivated Ganoderma lucidum or submerged fermentation of Ganoderma lucidum, the present invention proposes a method for the heterologous biosynthesis of Ganoderma lucidum triterpenoids by means of synthetic biology. Biosynthesis-related CYP genes are heterologously expressed in Saccharomyces cerevisiae cells, thereby achieving heterologous synthesis of Ganoderma lucidum triterpenes

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  • Method for heterologous biosynthesis of ganoderic acid through synthetic biological means
  • Method for heterologous biosynthesis of ganoderic acid through synthetic biological means
  • Method for heterologous biosynthesis of ganoderic acid through synthetic biological means

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction of yeast transformants

[0037] Construction of yeast strains expressing CYP genes includes:

[0038] Using the ganoderma lucidum cDNA library as a template, the coding sequence (CDS) fragments of each candidate gene were amplified by PCR;

[0039] Subsequently, each CDS fragment was recombined into the expression vector pRS426, and verified by sequencing. A series of recombinant plasmids pRS426HF-CYPs (s represent different CYP genes) were obtained;

[0040] Each recombinant plasmid was transformed into yeast cell YL-T3, thereby obtaining a series of yeast strains YL-T3-CYPs expressing different candidate CYP genes.

[0041] The primers for amplifying the ganoderma lucidum CYP gene cDNA fragment are shown in Sequence Table 1:

[0042] Table 1: List of primer sequences used to amplify Ganoderma lucidum CYP genes

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] F and R represent forward and reverse primers, respectively; lowercase lette...

Embodiment 2

[0061] Identification of Candidate CYP Genes and Functional Characterization Using Fermentation of Yeast Transformants

[0062] As mentioned above, we selected 82 candidate genes out of a total of 219 CYP genes in Ganoderma lucidum based on two principles, and completed the preliminary screening of 76 of them. The Gene IDs of these genes are: GL17567, GL17743, GL23374, GL21030, GL19267, GL23303, GL29510, GL15605, GL31771, GL31772, GL23109, GL20660, GL22911, GL19231, GL22909, GL330761, GL28 GL22480,GL24022,GL31754,GL24426,GL31403,GL23174,GL28081,GL29831,GL22657,GL31713,GL24902,GL17382,GL23363,GL24917,GL24889,GL21993,GL22087,GL21992,GL22088,GL24898,GL24883,GL24896,GL24382,GL24198,GL26139, GL21131,GL21663,GL31753,GL31777,GL15091,GL26850,GL31726,GL21057,GL29946,GL20766,GL20706,GL31717,GL31719,GL31723,GL23851,GL16778,GL23557,GL23927,GL23926,GL31718,GL31721,GL31722,GL28603,GL17412,GL31729, GL21090, GL31780, GL22978, GL21701, GL30595, GL30444, GL31768.

[0063] Fermentation of yeas...

Embodiment 3

[0068] Determination and Dynamic Accumulation Process of Fermentation Products of Yeast Transformants

[0069] 3.1.1) High performance liquid chromatography analysis method of fermentation product:

[0070] Instrument: Agilent 1200 Analytical HPLC; Chromatographic column: Agilent ZORBAX SB C18 reverse-phase chromatographic column (5um, 4.6x250mm)

[0071] Column temperature: 30°C; Flow rate: 1mL / min; Injection volume: 20μl;

[0072] Phase A: methanol (containing 0.1% acetic acid), phase B: pure water;

[0073] Gradient elution program: initial phase A 80%, phase B 20%; 0-30min, 80%-100% phase A; 30-50min, 100% phase A. The detection wavelength is 210nm.

[0074] 3.1.2) By comparing with the HPLC peak diagram of the fermentation product of the empty plasmid control strain, it was observed that when the recombinant plasmid containing CYP5150L8 was introduced into the yeast, the recombinant strain was significantly different from the control at 23.38min ( figure 2 ).

[007...

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Abstract

The invention discloses a method for heterologous biosynthesis of ganoderic acid through synthetic biological means. The method comprises the following steps: exploring cytochrome P450 enzyme (CYP) gene related to bio-synthesis of ganoderma triterpenes, and promoting heterologous expression of the gene in saccharomyces cerevisiae cells so as to achieve screening and identifying; and separating andpurifying a fermentation product of a yeast engineering strain, and conducting such analysis as mass spectrum, nuclear magnetic resonance and the like, so as to confirm 3-hydroxy-lanosta-8 and 24-dien-26-oci-acid (ganoderic acid Z). The method provided by the invention comprises a series of design and verification of exploring the bio-synthesis catalysis element (the gene) from the ganoderma triterpenes, fermentation phenotype priliminary screening and element function identifying, and finally, obtaining ganoderma triterpene substances of heterologous biosynthesis and the like, and the methodalso offers an example for heterologous production of other ganoderma triterpene active substances.

Description

technical field [0001] The invention relates to a technique in the field of bioengineering, in particular to a method for biosynthesizing ganoderma triterpenoids. Background technique [0002] Ganoderma lucidum (Ganoderma lucidum) is a traditional Chinese medicinal fungus, which has significant effects on adjuvant tumor therapy, anti-HIV, improving human immunity, and delaying aging (Sliva, 2004). These unique effects of Ganoderma lucidum are closely related to its intracellular physiologically active secondary metabolites, especially the triterpenoids of Ganoderma lucidum (Qin et al., 2016). At present, about 200 ganoderma triterpenoids have been isolated from Ganoderma lucidum (Baby et al., 2015), and they have different physiological functions. Tang et al., 2006; Chen et al., 2008), Ganoderma acid GS-2 can inhibit the replication of HIV virus (Sato et al., 2009). [0003] Cultivated Ganoderma lucidum has a long growth cycle, low content of triterpenoids and is easily af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12P33/00C12N15/53C12R1/865
CPCC12N9/0081C12N15/81C12P33/00C12Y114/15006
Inventor 肖晗王文方钟建江
Owner SHANGHAI JIAO TONG UNIV
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