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Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide-degrading enzymes

A polysaccharide degrading enzyme and genetic modification technology, applied in the field of lactic acid-utilizing bacteria that are genetically modified to secrete polysaccharide degrading enzymes, can solve the problems of high cost, limited carbohydrate source, low carbon-lactic acid conversion rate, etc., and achieve cost efficiency High, effective mixing of waste, pollution risk reduction effect

Inactive Publication Date: 2018-11-09
3PLW LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The methods of producing lactic acid from renewable resources described so far have many disadvantages, such as low carbon-to-lactic acid conversion, complex procedures requiring many individual steps, relatively high costs, and limited carbohydrate sources that can be used
Additionally, some methods are unfavorable due to the use of source materials that are of high value as human food

Method used

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  • Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide-degrading enzymes
  • Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide-degrading enzymes
  • Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide-degrading enzymes

Examples

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Effect test

preparation example Construction

[0165] Preparation of separated lactic acid enantiomers

[0166] In some embodiments, the reducing sugars of the organic waste (those reducing sugars originally found in the waste and those released by the action of polysaccharide degrading enzymes) can be fermented into lactic acid by microorganisms producing LA. In order to produce only one isolated lactic acid enantiomer, the LA-producing microorganism used produces only one of D-lactic acid and L-lactic acid enantiomer. The LA-producing microorganism can naturally produce only one enantiomer, or can be genetically modified to produce only one enantiomer, for example, by knocking out one or more enzymes involved in the synthesis of unwanted enantiomers.

[0167] In some embodiments, prior to lactic acid fermentation, the LA-utilizing bacteria are inactivated to avoid consumption of lactic acid produced during fermentation. For example, inactivation can be performed by increasing the temperature or changing the pH to a temperat...

Embodiment 1

[0211] Example 1-Construction of suitable vectors for genetically modified bacteria

[0212] Construction of Escherichia coli-Propionibacterium freudenreichii shuttle vector:

[0213] The constructed vector can be used in both Escherichia coli and Propionibacterium freudenreichii.

[0214] Prepare the pOWR3 vector as follows (the schematic diagram is shown in Figure 2A Middle): Propionibacterium replication protein sequence (SEQ ID NO.84) and chloramphenicol resistance gene (cmx (A) (SEQ ID NO: 85) and cml (from Corynebacterium striatum pT10 plasmid) A) (SEQ ID NO: 86)) was synthesized in tandem into a vector containing the E. coli origin of replication (ori) and the ampicillin resistance cassette (AmpR). This results in a ~5.6 kbp plasmid that can replicate in both E. coli and P. freudenreichii. The vector also includes the PB origin of replication sequence. The resulting pOWR3 vector is smaller than other known P. freudenreichii expression vectors (5.6 kbp vs. 6.2-8 kbp), whi...

Embodiment 2

[0219] Example 2—Construction of suitable expression cassettes and expression vectors for genetic modification of bacteria to express and secrete polysaccharides Degrading enzyme

[0220] Listed below are exemplary polysaccharide degrading enzymes (glucoamylase, cellulase, and hemicellulase (xylanase), signal peptides, and promoters used to generate expression cassettes used in expression vectors.

[0221] The following exemplary enzymes listed in Table 1 below are used for expression cassettes and expression vectors for genetically modified bacteria. Enzyme sequences were obtained from different sources (biological). The original signal peptide of each listed enzyme was removed and replaced with an exogenous (different) signal peptide.

[0222] Table 1-Exemplary polysaccharide degrading enzymes

[0223]

[0224]

[0225] The following promoter regions are used to construct various expression cassettes and expression vectors:

[0226] -PFREUD_04850 promoter from Propionibacterium fr...

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Abstract

Lactic acid (LA)-utilizing bacteria are provided, genetically modified to express and optionally secrete polysaccharide-degrading enzymes, such as cellulases, hemicellulases and amylases, and uses thereof. The polysaccharide-degrading enzymes are advantageous for processing organic waste so that the organic waste can be used as a substrate in industrial fermentation processes, particularly industrial production of discrete lactic acid enantiomer(s). Vectors and constructs useful for genetically modifying the LA-utilizing bacteria are also provided.

Description

Technical field [0001] The present invention relates to a dual-acting lactic acid (LA)-utilizing bacteria, which is genetically modified to secrete polysaccharide-degrading enzymes, such as cellulase, hemicellulase and amylase, for the treatment of organic waste to remove lactic acid present in the waste and Degradation of complex polysaccharides, thereby providing substrates for industrial fermentation processes, and producing various biochemicals, including specific lactic acid enantiomers. Background technique [0002] In recent years, since lactic acid can be used as a basic material in the manufacture of bioplastics, lactic acid fermentation, that is, the production of lactic acid from carbohydrate sources through microbial fermentation, has attracted interest. Lactic acid can be polymerized to form the biodegradable and recyclable polyester polylactic acid (PLA), which is considered a potential substitute for plastics made from petroleum. PLA is used in the manufacture of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/22C12N9/28C12N9/42C12N15/31C12N15/56C12N15/74
CPCC12N1/38C12N9/2411C12N9/2477C12P7/56C12N9/2437C02F3/34C02F2103/32C12P19/14
Inventor T·沙皮拉A·奥兰尼
Owner 3PLW LTD
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