Method of using lignocellulose for preparing DHA

A technology of lignocellulose and lignin, which is applied in the field of docosahexaenoic acid preparation, can solve problems such as complex process, high equipment requirements, and inability to interact with cell bodies, so as to meet market demand, wide source of raw materials, The effect of cheap raw material sources

Active Publication Date: 2018-11-27
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

The strategy of using fungal-derived enzyme preparations has insufficient efficiency and cost, mainly because the cellulase preparation needs to be prepared by microbial fermentation in a separate reactor, and the preparation process is different from the cellulose hydrolysis conditions, and the production steps are cumbersome Complexity, significant increase in manpower and material requirements and equipment investment costs make the saccharification process of lignocellulose uncompetitive in the market, so it is difficult to achieve large-scale industrial application of saccharification and DHA preparation technology based on fungal cellulase preparations
Invention patent 2015108721949 provides a method of directly using bagasse hydrolyzate to ferment Schizochytrium fungus. The substrate bagasse is hydrolyzed with dilute acid to obtain hydrolyzate instead of enzymatic hydrolysis, which has the potential risk of environmental pollution; in addition, due to Substances such as furfural in the hydro

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  • Method of using lignocellulose for preparing DHA

Examples

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Comparison scheme
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 2113813 to 2111293 nucleic acid sequence 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

The invention provides a process for producing DHA with lignocellulosic agricultural and forestry waste as a raw material in order to solve problems of industrial production of DHA in the prior art. The process includes the steps of pretreatment, saccharification, fermentation of schizochytrium limacinum, extraction of DHA and the like. The process adopts a combination of lignocellulosic biosaccharification and high-density schizochytrium limacinum fermentation; by adopting lignocellulosic biomass as the raw material, on the one hand, the production cost of DHA is significantly reduced; on theother hand, the problem of comprehensive utilization of the agricultural and forestry waste is also solved. In the process, a lignocellulose saccharification stage culture medium and a fermentation medium can be recycled so that water can be significantly saved, and the use of chemicals is reduced. In addition, the cellulose saccharification efficiency is 80-90%, the biomass of schizochytrium limacinum can reach 15-80g/L, and the production of DHA is 5-25g/L. Compared with the prior art, the process has the advantage that the production and yield of DHA are higher.

Description

technical field [0001] The invention relates to the field of microbial fermentation engineering, in particular to a method for preparing docosahexaenoic acid from lignocellulose raw materials. Background technique [0002] Docosahexaenoic Acid (DHA) is an essential polyunsaturated fatty acid that must be supplemented from the diet. DHA is an important structural lipid component of the central nervous system and retina. It has the functions of strengthening the brain, improving memory and vision, especially promoting the development and growth of brain cells in fetuses and infants, improving the memory of adolescents, and preventing and treating senile dementia. Traditionally, DHA is mainly derived from deep-sea fish oil, but due to the great impact of overfishing, environmental pollution, seasonal changes, etc., it is gradually replaced by microbial fermentation production. At the present stage, the industrial DHA production is mainly realized through the fermentation of Au...

Claims

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Application Information

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IPC IPC(8): C12P7/64C12P19/14C12P19/02C12R1/645
CPCC12P7/6427C12P19/02C12P19/14C12P2201/00C12P2203/00
Inventor 崔球刘亚君刘世岳宋晓金
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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