Engineered yeast for fermentation production of alpha-santalene and application of engineered yeast

A technology of yeast engineering and santalene, which is applied in the field of bioengineering, can solve the problems of low yield and limited application, and achieve the effects of broad application prospects, resource saving, and short cycle

Pending Publication Date: 2022-05-17
ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although santalene has many application prospects, its current low yield limits its application in different fields
The synthesis of plant-derived natural products by microorganisms is a very environmentally friendly and efficient way. There is no report on the production of santalene by Pichia pastoris cell factory

Method used

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  • Engineered yeast for fermentation production of alpha-santalene and application of engineered yeast
  • Engineered yeast for fermentation production of alpha-santalene and application of engineered yeast
  • Engineered yeast for fermentation production of alpha-santalene and application of engineered yeast

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: Construction of Pichia santalene recombinant strain Pa-1

[0044] The Int refers to the insertion site of the Pichia pastoris GS115 genome, and the used auxiliary plasmid Int-pTEF-tAOX1, Int-pGAP-tAOX1 consists of a general framework (ampicillin resistance gene expression cassette plus prokaryotic gene plasmid origin of replication ori), 500bp homology arms upstream and downstream of the site, and pTEF-BamHI-tAOX1 or pGAP-AatⅡ-tAOX1 (see the plasmid map image 3 , Figure 4 ); the helper plasmids HZP-sgRNA, HHP-sgRNA and HGP-sgRNA consist of a general framework, resistance gene expression cassette, sgRNA corresponding to the site, and BsaI restriction site, Z, H, and G represent bleomycin resistance sex gene, hygromycin resistance gene and G418 resistance gene (see plasmid map Figure 5 , Figure 6 , Figure 7 ); P is a plasmid (plasmid).

[0045] Starting from the helper plasmid Int2-pTEF-tAOX1 (preserved in the laboratory, Int2 left gene PAS_chr1-3_00...

Embodiment 2

[0060] Embodiment 2: Construction of Pichia santalene recombinant strain Pa-2

[0061] The expression cassettes of highly expressed genes tHMG1, IDI1, and ERG20 and the corresponding guide plasmids were respectively constructed, and they were sequentially transferred into the above-mentioned recombinant strain Pa-1, and the Pichia pastoris recombinant strain Pa-1-1 was obtained after successive rounds of transformation. , Pa-1-2 and Pa-2, the specific implementation is as follows:

[0062] (1) Construction of tHMG1 recombinant expression vector

[0063] Using the Saccharomyces cerevisiae genome as a template, design primers tHMG1-F / tHMG1-R to amplify the target gene tHMG1 (as shown in SEQID NO.2), and use the seamless cloning method to combine it with the vector Int12-pGAP- tAOX1 (Int12: left gene PAS_chr3_1202, right gene PAS_chr3_0618) was assembled and transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int12-pGAP-tHMG1-tAOX1.

[0064] The prime...

Embodiment 3

[0104] Embodiment 3: Construction of Pichia santalene recombinant strain Pa-3

[0105] Construct the expression cassette of the gene encoding the high-expression acetyl-CoA synthase ACS and the corresponding guide plasmid, electrotransform it into the above-mentioned recombinant strain Pa-2, and finally obtain the Pichia pastoris recombinant strain Pa-3, the specific implementation is as follows:

[0106] (1) Construction of the recombinant expression vector of the coding gene of acetyl-CoA synthase ACS

[0107] Using the acetyl-CoA synthase ACS plasmid stored in the laboratory as a template, design primers ACS-F / ACS-R to amplify the target gene ACS (as shown in SEQ ID NO.5), and use the seamless cloning method to combine it with BamHI The digested vector Int16-pTEF-tAOX1 (Int16: left gene PAS_chr3_1065, right gene PAS_chr3_1067) was assembled and transformed into Escherichia coli DH5α strain to obtain the recombinant vector Int16-pTEF-ACS-tAOX1.

[0108] The primer sequences...

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Abstract

The invention discloses a yeast engineering bacterium for fermentation production of alpha-santalene and application of the yeast engineering bacterium, and belongs to the field of bioengineering. Yeast is used as an original strain, and the yeast engineering bacterium for fermentation production of alpha-santalene is obtained through gene introduction construction. The engineered yeast strain can efficiently produce alpha-santalene, the shake flask fermentation yield of the engineered yeast strain is nearly 3.0 g/L, and the fermentation yield of a 1L fermentation tank can reach 15 g/L. The metabolic flux in the alpha-santalene biosynthesis pathway is improved through high expression of key rate-limiting enzyme in the metabolic pathway, enhancement of precursor supply and a multi-copy strategy, the alpha-santalene is efficiently generated by using the pichia pastoris, and the method has the characteristics of short period, environmental protection and resource saving. The engineered yeast strain for producing alpha-santalene at high yield has a wide application prospect.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a yeast engineering bacterium for fermenting and producing α-santalene and its application. Background technique [0002] Santalene, molecular formula C 15 h 24 , belonging to bicyclic sesquiterpenoids. There are two isomers of α-santalene and β-santalene. α-Santalene has a boiling point of 252°C (100.39kPa), and exists in the volatile oil of sandalwood. As one of the main components of sandalwood essential oil, α-santalene has a long-lasting aroma and is widely used in the flavor and fragrance industries. It also has pharmacological activities such as antibacterial, antioxidative and antitumor, and can be used as a valuable health medicine. [0003] Natural sandalwood oil mainly comes from the heartwood of sandalwood, and natural sandalwood essential oil is prepared by extraction methods such as organic reagent extraction and distillation, mainly by steam distillation, ether im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/52C12N15/53C12N15/54C12N15/60C12N15/61C12N15/81C12P15/00C12R1/84C12R1/865C12R1/645
CPCC12N9/88C12N9/0006C12N9/1085C12N9/93C12N9/90C12N15/815C12P15/00C12Y402/03082C12Y101/01034C12Y205/0101C12Y602/01001C12Y503/03002C12N2800/102
Inventor 连佳长左一萌程锦涛
Owner ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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