Method for producing oil rich in polyunsaturated fatty acids by using lignocellulose

An unsaturated fatty acid, lignocellulose technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem that the conversion efficiency cannot meet the requirements of industrial production, increase the amount or number of additions of enzymes, and prevent small fibers. In order to achieve the effect of cheap source of raw materials, reducing the proportion, and reducing the use of chemicals

Active Publication Date: 2022-04-15
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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AI Technical Summary

Problems solved by technology

However, since the activity and stability of exogenously added proteins will decrease with the progress of the saccharification process, the consumption of enzymes requires an increase in the amount or frequency of enzyme addition
In addition, these non-cellulosomal proteins are added to the saccharification system as free enzymes, which cannot interact with cellulosomes, and will significantly reduce the synergistic effect with other enzymes in the system, inevitably resulting in additional enzymes that are greater than the demand, resulting in The high cost of enzyme preparations
Not only that, this saccharification strategy that relies on the addition of exogenous free enzymes has complex processes, high equipment requirements, and conversion efficiency that cannot meet the requirements of industrial production.

Method used

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  • Method for producing oil rich in polyunsaturated fatty acids by using lignocellulose

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0051] Through seamless cloning, the tdk expression cassette (including the promoter of the gapDH gene) and the pyrF expression cassette (including the pyrF self-promoter) were cloned into the plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G.-Z., Yang, Y., Cui, Q., et al. (2013) A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum, PLoS One 8:e69032.) downstream of the antibiotic gene cat, And through the design of primers, NheI and XbaI restriction sites were added between tdk and pyrF expression cassettes, and EagI and MluI restriction sites were added downstream of pyrF for cloning of homology arm fragments, thereby constructing pHK-HR plasmid.

[0052] The cellulase Cel9K (exocellulase, encoded by the nucleic acid sequence 2113813 to 2111293 in the genome CP002416.1) in the celluloso...

Embodiment 2

[0058] Embodiment 2: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0059] The difference from Example 1 is that the polypeptide fragment II or polypeptide fragment I was linked to the 3' end of cellulosome endonuclease CelZ (SEQ ID NO: 15). The constructed recombinant strain is cultured to the mid-logarithmic phase in GS-2 medium with 5 grams per liter of cellulose or cellobiose as the carbon source, and can be used as a whole-bacteria enzyme preparation for biosaccharification of lignocellulose.

Embodiment 3

[0060] Embodiment 3: By the method of direct connection, construct the cellulase preparation based on Clostridium thermocellum cellulosome

[0061] Using the overlap extension polymerase chain reaction method, cellulosic exonuclease Cel9-48 (SEQ ID NO: 16) was combined with the sequence of type II adhesion module CohIIct (SEQ ID NO: 7) or type I of Clostridium thermocellum The sequence of the docking module DocIct (SEQ ID NO: 6) was directly connected, wherein the sequence of CohIIct or DocIct was connected to the 3' end of the Cel9-48 sequence to obtain the sequence of Cel9-48-DocIct or Cel9-48-CohIIct.

[0062] Using the Cel9-48-DocIct or Cel9-48-CohIIct sequence as the target sequence, using the MluI and EagI restriction sites to clone into the homologous recombination plasmid pHK-HR, using the lactate dehydrogenase gene clo1313_1160 as the targeted replacement sequence, The homologous recombination plasmid pHK-HR-cel9-48 was constructed. The upstream homology arm HR-up is...

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Abstract

Aiming at the problems existing in the prior art, the invention provides a process for preparing oil rich in polyunsaturated fatty acids by fermenting Mortierella alpina with lignocellulosic agricultural and forestry wastes as raw materials. The process includes the steps of pretreatment, saccharification, Mortierella alpina fermentation, oil extraction and the like. The fermentation stage of Mortierella alpina in the present invention adopts the method of gradually lowering the temperature, which can significantly increase the ARA output and reduce the proportion of saturated fatty acids in the oil, and the proportion of ARA in the total oil can reach 48%. Compared with the prior art, the process of the present invention adopts the cellulase preparation based on cellulosome-producing bacteria to realize the saccharification of lignocellulose, which significantly reduces the cost of carbon source and the enzyme cost in the saccharification stage, and the saccharification of cellulose Efficiency is 80%‑90%. In addition, the lignocellulose saccharification stage medium and fermentation medium can be recycled, which can significantly save water and reduce the use of chemicals, and has a significant effect of reducing waste water discharge and reducing costs.

Description

technical field [0001] The invention relates to the field of microbial fermentation engineering, in particular to a method for cultivating Mortierella alpina by using lignocellulose raw materials to produce oil rich in arachidonic acid and other polyunsaturated fatty acids. Background technique [0002] Mortierella alapina (Mortierella alapina) is a filamentous fungus of the genus Mortierella in the family Mucorales, which is widely distributed in soil, feces and other decaying organic substances. It is currently the fungus that produces arachidonic acid. The only strain with a formal safety assessment (GRAS). Because it can accumulate a considerable proportion of arachidonic acid (ARA or AA), Mortierella chrysogenum, which belongs to the same genus Mortierella, has received widespread attention and is recognized as an excellent strain for arachidonic acid production. Arachidonic Acid (ARA or AA for short) is an essential omega-6 long-chain polyunsaturated fatty acid that m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/6427C12P19/14C12P19/02C12R1/645
CPCC12P7/6427C12P19/02C12P19/14C12P2203/00C12P2201/00
Inventor 崔球刘亚君李仁民宋晓金张慧丹
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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