Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof

A technology for producing strains and high-yielding strains, applied in the field of modern biology, can solve problems such as restricting large-scale production, no effect, and inability to obtain transformation, and achieve the effects of low by-products, high yield, and stable genetic traits

Active Publication Date: 2016-09-07
SUZHOU BIOSYNTHETICA CO LTD +1
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  • Abstract
  • Description
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Problems solved by technology

The three methods have their own advantages and disadvantages. The animal and plant tissue extraction method restricts the production of coenzyme Q due to the supply cycle of raw materials and the source of the source. 10 mass production of
One of the problems often encountered in the modern biological breeding of traditionally bred microorganisms used for industrial fermentation is that conventional DNA transformation techniques for wild-type strains have a negative effect on those traditional breeding

Method used

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  • Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof
  • Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof
  • Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of expression plasmids

[0043] (a) Preserve pCL1920_pTrc in this laboratory ( www.synthesisgene.com ) and pMG160 (Inui, M, et al AEM 69 (2003) 725-733) as templates to construct Rhodobacter sphaeroides expression vector pBM03;

[0044] (b) Using E.coli W3110 as a template, primers UbiF-F and UbiF-R amplify the UbiF (Genbank: NC_000913.3) fragment; using the genome of coenzyme Q10 production strain-Rhodobacterium sphaeroides SZ as a template, using primer UbiH1 -F and UbiH1-R were amplified to obtain UbiH1 (Genbank: NC_007493, Rsp_1492), and primers UbiH2-F and UbiH2-R were used to amplify to obtain UbiH2 (Genbank: NC_007493, Rsp_1869). The corresponding primer series are shown in the sequence listing as SEQ ID NO : as shown in 1;

[0045] (c) The constructed expression plasmids are pBM03-UbiF, pBM03-UbiH1, and pBM03-UbiH2, respectively.

[0046] Both steps (a) and (c) were obtained by EZ fusion method (Generay GR6086). Rhodobacter sphaeroides SZ has be...

Embodiment 2

[0048] Construction of strain fst△(Rhodobacter sphaeroides 2.4.1ΔrshI)

[0049] (a) Using pK18mobSacB preserved in our laboratory as a vector, using the coenzyme Rhodobacter sphaeroides2.4.1 genome as a template, and using primers rshI-5'-F / R and rshI-3'-F / R, construct plasmid pK18mobSacB_rshI;

[0050] (b) Transform pK18mobSacB_rshI into the E.coli S17-1 host preserved in our laboratory to obtain E.coli S17-1 / pK18mobSacB_rshI;

[0051] (c) Transfer pK18mobSacB_rshI from E.coli S17-1 / pK18mobSacB_rshI to Rhodobacter sphaeroides and Fst bacteria through conjugation transfer experiment, and obtain the zygote of single exchange strain Fst_rshI by screening with nalidixic acid + resistance marker. For detailed conjugation transfer methods, see Porter SL, et al Methods Enzymol 423:392-413 (2007).

[0052] (d) A non-resistant double-crossover strain-Rhodobacterium sphaeroides Fst△ was obtained by screening with 20% sucrose. After using rshI-5'-F / rshI-3'R primers to amplify the geno...

Embodiment 3

[0055] Preparation of Fst△ and SZ Competent

[0056] (a) Separate 20 ul from the glycerol tubes containing Fst△ bacteria and SZ bacteria, streak on LB plates, and culture overnight at 34°C in the dark;

[0057] (b) Pick a single bacterium into a 4ml LB test tube, and activate overnight at 34°C in the dark until the cell OD ≤ 1.0;

[0058] (c) Transfer 4ml of the bacterial solution in (b), inoculate into a 500ml shake flask containing 100ml LB medium, culture at 34°C, 220rpm, in the dark for 4-6h;

[0059] (d) But when the OD of the above-mentioned culture solution reaches 0.6, collect the bacteria by centrifuging at 6000rpm for 3min;

[0060] (e) Wash the thalline 3 times with sterile water subsequently, centrifuge at 6000rpm, 3min;

[0061] (f) Add 0.5-1ml of the E2 preparation solution to the prepared cells to suspend the cells, divide each 80ul cells into eppendrof tubes, and store at -80°C for later use.

[0062] Rhodobacter sphaeroides SZ in step (a) is a coenzyme Q10 ...

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Abstract

The invention discloses a method for reducing or eliminating a by-product D in a coenzyme Q10 producing strain SZ. According to the method, the gene of 5-demethoxy panthenol hydroxylase is expressed in the coenzyme Q10 producing strain SZ, so that the accumulation of the by-product D is reduced or eliminated, wherein the producing strain SZ is classified and named as rhodobacter sphaeroides, which is preserved in China General Microbiological Culture Collection Center on March 3, 2016, with preservation number of CGMCC NO. 12177. The invention also discloses a coenzyme Q10 high-yield strain SF and an application thereof, wherein the high-yield strain SF is classified and named as rhodobacter sphaeroides, which is preserved in China General Microbiological Culture Collection Center on March 3, 2016, with preservation number of CGMCC NO. 12178. The high-yield strain SF is obtained by expressing the gene of the 5-demethoxy panthenol hydroxylase in the producing strain SZ; and with the application of the high-yield strain SF, the yield of produced coenzyme Q10 reaches 2g/L or above; therefore, the high-yield strain SF has a broad application prospect.

Description

technical field [0001] The invention relates to the technical field of modern biology, in particular to a 10 A method for reducing or eliminating by-product D in the production strain SZ, a coenzyme Q10 high-yielding strain and applications thereof. Background technique [0002] coenzyme Q 10 (Coenzyme Q 10 , CoQ 10 ), the chemical name is: 2,3-dimethoxy-5-methyl 6-decisopentenylbenzoquinone, and the molecular formula is C 59 h 90 o 4 , the relative molecular weight is 863, the melting point is 48-50 ℃, it is orange-yellow crystal at room temperature, odorless and tasteless. Soluble in chloroform, benzene, carbon tetrachloride, soluble in acetone, petroleum ether and ether, slightly soluble in ethanol, insoluble in water and methanol. It is easy to decompose into reddish under light, and is relatively stable to temperature and humidity. [0003] coenzyme Q 10 It is a fat-soluble quinone compound, which was first discovered in 1957. Due to its existence in human orga...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/66C12R1/01
Inventor 胡志浩王庆军范俊英张瑞萍韩祎君王欣彤施明雨王绪州
Owner SUZHOU BIOSYNTHETICA CO LTD
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