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Construction body and method for improving fatty alcohol yield in cyanobacteria

A construct and cyanobacteria technology, applied in the field of fatty alcohol production, can solve the problems of increased microbial toxicity, volatility, and high cost

Active Publication Date: 2013-03-06
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A technical route for the preparation of bioethanol has been developed, and large-scale industrial production of bioethanol can be realized; however, as a fuel, ethanol has some defects: (1) low energy density; (2) easy to volatilize; (3) its soluble Some problems caused by water, such as increased toxicity to microorganisms during fermentation, high cost of removing the water phase during distillation and separation, and corrosion of pipelines during transportation

Method used

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  • Construction body and method for improving fatty alcohol yield in cyanobacteria
  • Construction body and method for improving fatty alcohol yield in cyanobacteria
  • Construction body and method for improving fatty alcohol yield in cyanobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Construction of a vector for expressing fatty acyl-CoA synthetase

[0107] In order to increase the expression level of fatty acyl-CoA synthetase in cyanobacteria, the plasmid pGQ7 carrying and expressing slr1609 gene was constructed as follows.

[0108]With 1609NdeI (SEQ ID NO: 8, 5'-TAC ATA TGG ACA GTG GCC ATGGCG CTC AAT-3') and 1609R (SEQ ID NO: 9, 5'-CCC TCG AGA AAC ATTTCG TCA ATT AAA TGT T-3' ) as primers, using the cyanobacterium PCC6803 genomic DNA as a template for PCR amplification, and according to the manufacturer's instructions, the PCR amplification product was cloned into the pMD18-T vector (Takara, Catalog No.: D101A) to obtain plasmid pGQ3. After sequencing verification, the plasmid pGQ3 was digested with NdeI (Takara, Catalog No.: D1161A) and XhoI (Takara, Catalog No.: D1094A), and a DNA fragment of about 2.1 kb was recovered. In addition, plasmid pET21b (Novagen) was digested with NdeI (Takara, Catalog No.: D1161A) and XhoI (Takara, Catalog...

Embodiment 2

[0109] Example 2: Detection of the activity of fatty acyl-CoA synthetase

[0110]

[0111] To determine whether the plasmid pGQ7 can express a functional fatty acyl-CoA synthetase, as described by Hosaka et al., 1979, the activity of the protein expressed by the plasmid pGQ7 was measured based on the above several coupling reactions. The specific reaction system is as follows: Tris-HCl (pH7.4) 0.1mM, dithiothreitol 5mM, TritonX-100 1.6mM, ATP 7.5mM, magnesium chloride 10mM, oleic acid 0.25mM, coenzyme A (CoA) 1mM, phosphoenol Potassium pyruvate (PEPK) 0.2 mM, NADH 0.15 mM, adenylate kinase 11 U, pyruvate kinase 9 U, lactate dehydrogenase (LDH) 9 U, purified protein expressed by plasmid pGQ7 (ACSL) 1.8 mM. Finally, enzyme activity was determined by measuring the light absorbance of NADH at 340 nm. Assay results show that the protein expressed by plasmid pGQ7 has fatty acyl-CoA synthetase activity, and its kcat value (the molar amount of substrate that can be converted by ea...

Embodiment 3

[0112] Example 3: Construction of vectors for gene knock-in and gene knock-out

[0113] In order to confirm the role of fatty acyl-CoA synthetase in the production of fatty alcohols in cyanobacteria, and to confirm that the production of fatty alcohols in cyanobacteria can be increased by increasing the expression of the enzyme, the following construction for the fatty alcohols driven by the psbA2 promoter Acyl-CoA synthetase gene (slr1609 gene) is integrated into the vector pGQ49 of cyanobacteria genome, and the vector pGQ17 is used to knock out endogenous fatty acyl-CoA synthetase gene (slr1609 gene) in cyanobacteria.

[0114] 1. Construction of vector pXT68

[0115] Using Synechocystis PCC6803 genomic DNA as a template, Pd1-2-f (SEQ ID NO: 14, 5'-CAC AT A GAT CT G CCA GTT GAG GT-3′) and Pd1-2-r (SEQ ID NO: 15, 5′-GGG CAT ATG GTT ATA ATT CCT TAT GTA TTT G-3') primers for PCR amplification, and according to the manufacturer's instructions, the PCR product obtained was cl...

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PUM

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Abstract

The invention relates to a construction body, comprises a carrier of the construction body, comprises the construction body or cyanobacteria converted by the carrier, and relates to a method for improving fatty alcohol yield in cyanobacteria, wherein the cyanobacteria is modified to be capable of producing fatty alcohol. The invention also relates to a new method for producing fatty alcohol in cyanobacteria.

Description

technical field [0001] The invention relates to the fields of renewable energy and biomass energy. In particular, the present invention relates to constructs for increasing the production of fatty alcohols in cyanobacteria, vectors comprising said constructs, cyanobacteria comprising said constructs or transformed with said vectors, and increasing in cyanobacteria A method of fatty alcohol production wherein the cyanobacteria have been engineered to produce fatty alcohol. The invention also relates to novel methods for the production of fatty alcohols in cyanobacteria. Background technique [0002] At present, energy issues and environmental issues are gradually becoming prominent factors restricting sustainable economic and social development. The application of renewable biofuels is considered to be an effective way to solve these two problems. A technical route for the preparation of bioethanol has been developed, and large-scale industrial production of bioethanol can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/52C12N15/53C12N1/13C12P7/02C12R1/89
CPCC12P7/04C12N9/00C12Y203/01086C12N9/1029
Inventor 吕雪峰高倩倩王纬华赵辉
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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