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Method for synthesizing biodiesel with microorganisms in vivo

A technology of synthetic biology and microorganisms, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effect of reducing costs and improving synthetic capabilities

Active Publication Date: 2011-05-25
青岛生物能源与过程研究所
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Problems solved by technology

However, in this achievement of the Alexander Steinbuchel team, although the microbial synthesis of biodiesel has been realized for the first time, it is still necessary to provide E. coli with long-chain fatty acids from exogenous sources; Atoms are provided by exogenous fatty acids, so de novo synthesis of biodiesel in real microorganisms has not yet been achieved

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  • Method for synthesizing biodiesel with microorganisms in vivo
  • Method for synthesizing biodiesel with microorganisms in vivo
  • Method for synthesizing biodiesel with microorganisms in vivo

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Embodiment 1

[0031] A) clone atfA, fadD, pdc and adhB genes on pET28b vector

[0032] (1) Plasmid pXL67 is obtained by cloning atfA gene in pET28b vector through NdeI and SpeI; and plasmid pXL72 is obtained by cloning fadD gene in pCR-blunt vector; pYL6 and pYL5 are obtained by cloning pdc and adhB genes in pMD18 respectively -T vector obtained.

[0033] (2) There is an XbaI site upstream of the SpeI site of plasmid pYL6, which will affect the subsequent DNA molecular cloning operation and needs to be eliminated. First, pYL6 was completely digested with XbaI, then the cohesive ends were blunted with T4DNAPolymerase, and finally the DNA fragments were self-ligated to transform Escherichia coli. Obtained clones, extracted plasmids confirmed by sequencing that the XbaI site has been eliminated, this plasmid is pXT1 ( figure 1).

[0034] (3) Plasmids pXL72, pXT1 and pYL5 were digested with NdeI and SpeI, and fadD, pdc and adhB gene fragments were excised and recovered respectively.

[0035...

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Abstract

The invention relates to a method for synthesizing biodiesel with microorganisms in vivo, which is realized in escherichia coli. The method comprises the following steps of: respectively cloning an acinetobacter wax fat synthetase gene atfA, an escherichia coli acyl coenzyme A synthetase gene fadD, a zymomonas mobilis pyruvate decarboxylase gene pdc and an alcohol dehydrogenase gene adhB gene on a pET expression vector in series to obtain a plasmid pXT11; transferring the plasmid pXT11 into escherichia coli containing a plasmid pMSD8 and a plasmid pMSD15 to obtain an escherichia coli stain XT31; and generating biodiesel by the escherichia coli stain XT31. The method realizes the microbial transformation from sugar to the biodiesel, lays the foundation for biologically preparing the biodiesel from lignocelluloses, and radically solves the problem of insufficient raw material supply in biodiesel preparation. A step of esterifying methanol is not needed in the technology, thereby the cost is greatly reduced and the problems that glycerol as a side product is formed, and the like are avoided.

Description

technical field [0001] The invention belongs to the fields of renewable energy and biomass energy, and in particular relates to a method for synthesizing biodiesel from scratch in microorganisms. Background technique [0002] Biodiesel is mainly prepared from animal and vegetable fats and oils through transesterification process catalyzed by chemical method or enzymatic method. The main bottleneck problems faced by this production method are the shortage of production raw materials, the use of methanol and the removal of glycerin by-products, etc., and the most important problem is the insufficient supply of production raw materials. At present, the main oil resources (such as soybean oil, rapeseed oil, etc.) used in the industry to prepare biodiesel are also necessary for human life, while a large number of oil plants are planted to provide raw materials, and there are insufficient arable land and seasonal climate restrictions. question. Microalgae oil is considered to be...

Claims

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Application Information

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IPC IPC(8): C12P7/64C12N15/70C12N1/21C12R1/19
CPCY02E50/13Y02E50/10
Inventor 吕雪峰谈晓明段仰凯蔡轲朱至
Owner 青岛生物能源与过程研究所
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