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Construction method for high-yield sclareol strain

A technology of sclareol and its construction method, which is applied in the field of construction of high-yield sclareol strains, can solve problems such as not fully satisfying industrialized production, long growth cycle of plant sclareol, etc., and achieve the effect of high-efficiency production

Inactive Publication Date: 2013-11-13
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, most of the production of Sclareol is based on the extract of Sclareus inflorescences and stems and leaves after oil extraction. The disadvantage is that the growth cycle of the plant Sclareus is long, and it is limited by land, environment and climate. Factors, can not fully meet the needs of industrial production

Method used

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  • Construction method for high-yield sclareol strain
  • Construction method for high-yield sclareol strain
  • Construction method for high-yield sclareol strain

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Experimental program
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Embodiment 1

[0029] Whole Gene Synthesis of LPP Synthase and Sclareol Synthase

[0030] The protein sequences of LPP synthase and sclareol synthase of the plant Sclareus are derived from the patent (WO.Pat.NO.2009101126-A1) written by Schalk of Fumenisher Co., Ltd., Geneva, Switzerland. Utilize codon-optimized sites ( http: / / www.jcat.de / Start.jsp ), the genes of LPP synthase and sclareol synthase were designed according to the codon preference of Saccharomyces cerevisiae, and the optimally designed gene sequences were different from those in the Schalk patent, with differences of 23.92% and 23.50% respectively , see sequence 1 and sequence 2 for the specific sequence. The whole gene synthesis of LPP synthase and sclareol synthase was carried out by TaKaRa Company, and the numbers are CBG747 and CBG748 respectively.

[0031]The specific process of whole gene synthesis is as follows: First, synthesize 50-100nt single-stranded small fragment DNA according to the gene sequence, and splicing...

Embodiment 2

[0034] Production of Sclareol by Recombinant Strain S2

[0035] Recombinant strain S2 is a strain that co-overexpresses LPP synthase and sclareol synthase and overexpresses fusion FPP synthase and GGPP synthase, see figure 1 (2), its construction process is as follows. First, using the Saccharomyces cerevisiae genome or plasmid as a template, clone promoters (TEF1p-1, TDH 3p-6 and TPIp-1), terminators (TDH2t, pYX212t and FBA1t-1) and gene sequences (LPPs, Tps, BTS1 and ERG20), detailed information is shown in Table 1; secondly, the cloned DNA fragments were connected by fusion PCR method (Yue et al., 2004, Fungal genetics and biology. 41: 973-981). Among them, the fusion PCR method is divided into two rounds of PCR reactions, the first round is according to the combination of fragments in Table 2, a: 1 (35ng), 2 (450ng), 3 (100ng) and 6 (35ng); b: 3 (40ng ), 6(140ng), 7(500ng) and 8(40ng); c: 15(40ng), 16(140ng), 17(140ng), 18(100ng) and 1(40ng), respectively added to the PC...

Embodiment 3

[0067] Production of Sclareol by Recombinant Strain S3

[0068] Recombinant strain S3 is a strain that co-overexpresses truncated LPP synthase and truncated sclareol synthase and overexpresses fusion FPP synthase and GGPP synthase, see figure 1 (3), its construction process is basically the same as that of strain S2, the difference is that LPP synthase and sclareol synthase are truncated genes that remove the N-terminal part of the sequence, wherein the sequence length of the N-terminal truncated Referring to Schalk's patent (WO.Pat.NO.2009101126-A1), LPP synthase and sclareol synthase are truncated by 63aa and 50aa, respectively.

[0069]First, using the Saccharomyces cerevisiae genome or plasmid as a template, clone promoters (TEF1p-1, TDH3p-6 and TPIp-4), terminators (TDH2t, pYX212t and FBA1t-1) and gene sequences (tLPPs, tTps, BTS1 and ERG20 ), detailed information is shown in Table 4; secondly, the cloned DNA fragments were connected, and the method of fusion PCR was ado...

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Abstract

The invention discloses a method for constructing a high-yield sclareol engineering strain by a synthetic biology methdo, and an application thereof. The construction method for the strain relates to express plant sourced key enzymes for synthesizing sclareol, wherein the key enzymes comprise a pyrophosphoric acid ladanum enediol ester synthase and a sclareol synthase; and overexpress a combination of one or more yeast endogenous enzyme-based genes, wherein the enzyme-based genes comprise a key enzyme of a mevalonic acid approach-(hydroxymethyl) coenzyme A reductase, and two enzymes of an isopentenylpyrophosphate approach-a farnesyl pyrophosphate synthase and a geranylgeranyl diphosphate synthase. The method can efficiently produce sclareol, with a shake flask fermentation level reaches 9 mg / l, and has industrial application prospects.

Description

technical field [0001] The invention relates to a method for constructing a high-yield sclareol strain. More specifically, the present invention relates to methods for the production of sclareol by eukaryotic microorganisms. Background technique [0002] Isoprenoids, also known as terpenes or terpenoids, are the most diverse compounds that exist widely in nature. More than 40,000 different terpenoids have been isolated from plants, animals and microorganisms. out (Rohdich et al., 2005, Biochem Soc Trans. 33:785-791). The rich and diverse structures of these compounds provide a good compound screening library for solving human health and social problems, and some compounds have been widely used in medicine, health food, spices, agricultural chemicals, etc. (Misawa, 2011, Curr Opin Biotechnol.22 : 627-633). However, the yield of such compounds in natural hosts is extremely low, which largely limits the industrial production and application of such compounds. At present, on...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/54C12N15/53C12P7/02C12R1/865
Inventor 赵宗保杨薇
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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