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Yarrowia lipolytica genetically engineered bacterium for producing limonene and application of yarrowia lipolytica genetically engineered bacterium

A technology of Yarrowia lipolytica and genetically engineered bacteria, applied in the field of genetic engineering, can solve problems such as inability to meet, and achieve the effects of reducing production costs, increasing yield, and reducing waste

Active Publication Date: 2020-11-24
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, considering that the method of extracting limonene from waste citrus peel is affected by seasonality and cannot meet the demand, the production of limonene by microbial fermentation has attracted more and more attention.
[0005] However, there is no report on the synthesis of limonene from xylose and lignocellulose hydrolyzate using Yarrowia lipolytica cells as the chassis

Method used

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  • Yarrowia lipolytica genetically engineered bacterium for producing limonene and application of yarrowia lipolytica genetically engineered bacterium
  • Yarrowia lipolytica genetically engineered bacterium for producing limonene and application of yarrowia lipolytica genetically engineered bacterium
  • Yarrowia lipolytica genetically engineered bacterium for producing limonene and application of yarrowia lipolytica genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056]Example 1: Construction of xylose utilization metabolic pathway in Yarrowia lipolytica

[0057] (1) Since Yarrowia lipolytica itself can only metabolize a small amount of xylose, in order to use xylose to synthesize limonene, this example introduces the optimized xylose reductase gene XR and xylitol dehydrogenase gene XDH derived from Scheffersomyces stipites , and overexpressed the xylose assimilating enzyme gene XK from Yarrowia lipolytica.

[0058] (2) The optimized xylose reductase gene XR derived from Scheffersomyces stipites was constructed into the plasmid pHR_AXP_hrGFP through double restriction sites PteI and NheI to obtain the plasmid pHR_AXP_XR. The optimized xylitol dehydrogenase gene XDH derived from Scheffersomyces stipites was constructed into plasmid pHR_XPR2_hrGFP through double restriction sites PteI and NheI to obtain plasmid pHR_XPR2_XDH. The xylose assimilating enzyme gene XK from Yarrowia lipolytica was constructed into the plasmid pHR_A08_hrGFP th...

Embodiment 2

[0064] Example 2: Construction of limonene synthesis pathway in Yarrowia lipolytica

[0065] (1) The optimized sequence of the gene LS derived from Agastache rugosa and the optimized sequence of the gene NDPS1 derived from tomato (Solanum lycopersicum) were respectively constructed into plasmid pINA1312 through restriction site PmlI to obtain plasmids pINA1312-LS and pINA1312-NDPS1.

[0066] (2) The expression cassette of the LS gene in the plasmid pINA1312-LS obtained in step (1) was connected to the plasmid pINA1312-NDPS1 obtained in step (1) through the restriction site StuI to obtain the plasmid pINA1312LN containing the genes LS and NDPS1.

[0067] (3) The expression cassette of the LS gene in the plasmid pINA1312-LS obtained in step (1) is connected to the plasmid pINA1312LN obtained in step (2) through the restriction site ClaI to obtain two copies of the gene LS and one copy of the gene LS. Plasmid pINA1312LLN of the gene NDPS1.

[0068] (4) The plasmid pINA1312LLN o...

Embodiment 3

[0071] Example 3: Mevalonate (MVA) Pathway Optimization in Yarrowia lipolytica

[0072] (1) The plasmid pINA1269-HMG1-ERG12 was linearized and then transformed into the strain YBX07 obtained in Example 2 by homologous recombination to obtain strain YBX08.

[0073] (2) The obtained recombinant strain YBX08 was subjected to shake-flask fermentation, and the specific shake-flask fermentation method was the same as the fermentation method of strain YBX07 in YPX medium in Example 2. After the fermentation, the limonene production, dry cell weight (DCW) and limonene yield of the strain YBX08 were measured respectively according to the standard limonene assay method. The results are shown in figure 2 c.

[0074] From figure 2 It can be seen in C that strain YBX08 can use xylose as a carbon source to synthesize limonene, and the limonene production is higher than that of strain YBX07 in YPX medium.

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Abstract

The invention discloses a yarrowia lipolytica genetically engineered bacterium for producing limonene. A method comprises the following steps: introducing an optimized xylose reductase gene XR, an optimized xylitol dehydrogenase gene XDH and an optimized xylose assimilation enzyme gene XK into chromosomes of uracil and leucine auxotrophic yarrowia lipolytica to construct a yarrowia lipolytica genetically engineered bacterium YBX06; transforming a recombinant vector containing two copy-optimized limonene synthase genes LS and one copy-optimized neroli diphosphate synthase 1 gene NDPS1 into theyarrowia lipolytica genetically engineered bacterium YBX06, and performing construction to obtain the yarrowia lipolytica genetically engineered bacterium YBX07. The yarrowia lipolytica genetically engineered bacterium constructed by the invention can utilize lignocellulose hydrolysate as a raw material and synchronously utilize xylose and glucose as a mixed carbon source to produce the limonene through biological fermentation, so that the yarrowia lipolytica genetically engineered bacterium has a good application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Yarrowia lipolytica genetically engineered bacterium which can utilize xylose and lignocellulose hydrolyzate to high-yield limonene and an application thereof. Background technique [0002] Lignocellulosic biomass and agricultural waste produced through photosynthesis on the earth can reach 100 billion tons every year, which also makes this biomass the most abundant renewable resource on the earth. Efficient use of lignocellulose is essential to reduce the demand for energy and food. Xylose is the second most abundant monosaccharide after glucose in lignocellulosic hydrolysates, accounting for nearly 35% of all monosaccharides. However, due to the inhibitory effect of carbon catabolites, most microorganisms cannot efficiently utilize lignocellulose hydrolyzate to metabolize xylose, which largely limits the application of lignocellulose. Due to the high cost of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12N15/60C12N15/52C12P5/00C12R1/645
CPCC12N9/0006C12N9/88C12N9/1085C12N9/1205C12N9/00C12Y101/01307C12Y101/01009C12Y402/0302C12Y101/01034C12Y205/01028C12Y207/01036C12N15/52C12N15/815C12P5/002Y02E50/10
Inventor 韦柳静钟驭涛花强姚丰吕预备
Owner EAST CHINA UNIV OF SCI & TECH
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