WO2022062088A1 - Bactérie génétiquement modifiée pour transformer une biomasse contenant de la lignine pour synthétiser de la vanilline et son utilisation - Google Patents

Bactérie génétiquement modifiée pour transformer une biomasse contenant de la lignine pour synthétiser de la vanilline et son utilisation Download PDF

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WO2022062088A1
WO2022062088A1 PCT/CN2020/126908 CN2020126908W WO2022062088A1 WO 2022062088 A1 WO2022062088 A1 WO 2022062088A1 CN 2020126908 W CN2020126908 W CN 2020126908W WO 2022062088 A1 WO2022062088 A1 WO 2022062088A1
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lignin
vanillin
genetically engineered
transforming
synthesize
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朱道辰
孙建中
朱彬
许令侠
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江苏大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group

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  • the invention relates to the use of molecular biology technology to obtain Bacillus ligninophilic genetically engineered bacteria (hereinafter referred to as genetically engineered bacteria) capable of accumulating a large amount of vanillin by reconstructing the metabolic pathway of Bacillus ligninophilic strains, so as to realize the transformation of lignin-containing bacteria into
  • a method for converting biomass or industrial lignin and their depolymerization products to synthesize vanillin belongs to the field of biotechnology.
  • Vanillin (Vanillin, C 8 H 8 O 3 ), scientific name 3-methoxy-4-hydroxybenzaldehyde, also known as vanillin, is the world's largest and most widely used broad-spectrum high-grade spice. Used in food, daily chemical, rubber, plastic and pharmaceutical industries.
  • vanillin Chemical synthesis of vanillin is the main source of vanillin in the market, and the annual output has reached 20,000 tons.
  • chemically synthesized vanillin is low in cost and large in scale, it is easy to cause environmental pollution and cannot meet people's expectations for natural food. Require.
  • vanilla is mainly obtained through the extraction of vanilla beans.
  • due to the need for manual pollination in the cultivation of vanilla it is labor-intensive and difficult to grow on a large scale.
  • the planting volume of vanilla is only about 2000-3000 tons per year, the output of extracted natural vanillin is only about 20 tons, and the market price is as high as 3200 US dollars / kg.
  • consumers' demand for natural vanillin has been increasing, and some foreign (such as Europe) legislative bodies have recommended the use of natural vanillin in food, but the method of extracting from plant tissue has low yield and high cost , resulting in a shortage of natural vanillin.
  • Microbial degradation and conversion of lignin has the advantages of simple conversion process, high yield, low energy consumption, and product safety.
  • the U.S. Food and Drug Administration stipulates that "natural flavors must be products derived from plant or animal raw materials through physical, enzymatic or microbial processing.” Therefore, microbial transformation and synthesis of vanillin will be an important trend in future development.
  • lignin As the main source of the most abundant natural aromatic compounds in the world, lignin has not been valued and utilized for a long time. How to convert lignin components into high-value products is a feasible way to improve the comprehensive utilization of depolymerized lignin.
  • the lignin depolymerization product contains a large amount of aromatic compounds such as ferulic acid, cinnamic acid, vanillic acid, vanillin and coumaric acid.
  • the conversion of aromatic compounds in lignin into specific single aromatic compounds by biological methods has important practical significance for realizing high-value utilization of lignin, and is also a requirement for realizing the sustainable development of agriculture and forestry.
  • Rhodococcus jostii RHA045 can use wheat straw and glucose as raw materials to produce vanillin, and can reach a yield of 96 mg/L after 6 days of fermentation (Paul D.Sainsbury, et al. ACS chemical biology, 2013, 8 , 2151-2156).
  • this strain cannot use lignin as a single carbon source. Using alkaline lignin as a carbon source for fermentation for 48-72 hours, only 1.0-1.3g/L of vanillin is obtained. Obviously, this strain is not suitable for using lignin to produce aroma. Lansu.
  • the long fermentation time of 6 days also affects the efficiency of vanillin production, so it is difficult to realize the production of vanillin converted from lignin.
  • Bacillus ligninophilus L1 is an alkalophilic and salt-tolerant extremophile microorganism screened in deep-sea sediments. It can survive in the environment of pH 7-11, and L1 has strong growth adaptability (10-50°C, pH 6.0 -11.0, 0-10%NaCl, optimum growth pH 9), it is one of the most alkali-tolerant strains among the currently known lignin-degrading bacteria.
  • vanillin and vanillic acid are common intermediates of ⁇ -aryl ether, biphenyl, diarylpropane and ferulic acid, and account for up to 30% of the aromatic products of L1 depolymerized alkaline lignin.
  • vanillin is one of the most ideal target products for biodepolymerization and conversion of lignin. Vanillin can be converted to vanillic acid by vanillin dehydrogenase. Therefore, knocking out the vanillin dehydrogenase gene and blocking the degradation of vanillin can achieve a large accumulation of vanillin.
  • the technical problem to be solved by the present invention is to provide an efficient method for preparing vanillin by transforming lignin biomass and its depolymerization products by constructing genetic engineering strains.
  • the purpose of the present invention is to disclose a method for converting lignin, including various industrial lignin and paper-making black liquor, into a high value-added aromatic compound vanillin.
  • the method of the present invention solves the problem of high-valued lignin. Using the bottleneck, the biotransformation of vanillin has been realized, which has important industrial value.
  • the present invention discloses a genetically engineered bacterium that transforms lignin-containing biomass to synthesize vanillin.
  • the bacterium is named Bacillus ligniniphilus L1-vdh, which has been deposited in The General Microbiology Center of China Microbial Culture Collection Management Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China.
  • the proposed classification name is Bacillus ligniniphilus, and the deposit number is CGMCC No.19226.
  • lignin depolymerization mixture The industrial lignin or the pretreated lignin-containing pretreatment liquid is prepared by one of the thermal cracking method, the enzymatic depolymerization method, the microwave, the metal catalyst cracking method, and the alkaline hydrolysis method. This kind of treatment can achieve the purpose of depolymerizing lignin, and the pH of the depolymerization reaction solution is adjusted to neutral for use.
  • step (1) the lignin aqueous solution not higher than 10% (v/v) is thermally cracked at a temperature of 250-450° C. for 5-30 minutes in a reactor.
  • the pressure is controlled within 30 MPa.
  • step (1) the laccase is dissolved in acetate buffer (pH 4) at a concentration of 0.1g/L, and then the laccase solution is dissolved at a concentration of 1:1 (v/v) The ratio was mixed with 10% lignin solution and reacted at 30°C for 12 hours.
  • step (1) adding the sodium hydroxide solution of not more than 10% (g/v) to lignin (the lignin content in the solution is not more than 10g/L), at 80-100 ° C Process 1-6h.
  • the metal catalyst cracking method described in step (1) using metal catalyst (nickel, titanium, zinc, etc.) as a catalyst, treating in a 10% lignin solution system at 100-300° C. for 0.5-1 h. Dissolve lignin in 2-10% NaCl solution at a concentration of 1-10% (g/L), then react in a reactor at 60-200° C. for 1-3 hours and collect the filtrate for later use.
  • metal catalyst nickel, titanium, zinc, etc.
  • the method is to supplement glucose when the glucose is lower than 5g/L, stop the fermentation when the fermentation OD value is above 30, and then replace the fermentation broth with MM63 medium with lignin depolymerization product as a single carbon source, and continue to cultivate at 37 °C for 24h , the pH was adjusted to about 9 by NaOH and HCl, and the amount of vanillin synthesis was detected by HPLC.
  • the 2216E medium in step (2) has the following components: peptone 5g/L, yeast powder 1g/L, ferric citrate 0.1g/L, sodium chloride 19.45g/L, magnesium chloride 5.97g/L, sodium sulfate 3.24g/L , calcium chloride 1.8g/L, sodium carbonate 0.16g/L, potassium bromide 0.08g/L, strontium chloride 0.034g/L, boric acid 0.022 g/L, sodium silicate 0.004g/L, sodium fluoride 0.0024 g/L, sodium nitrate 0.0016g/L, sodium dihydrogen phosphate 0.008g/L.
  • composition of the fermentation medium described in step (2) is as follows: enzymatic hydrolysis soybean meal powder 50g/L, corn steep liquor powder 30g/L, glucose 10g/L, FeSO 4 0.1g/L, MgSO 4 0.5g/L , phosphate buffer 50mM.
  • the components of the MM63 medium described in step (2) are as follows: 100 mM KH 2 PO 4 , 75 mM KOH, 15 mM (NH 4 ) 2 SO 4 , 1 mM MgSO 4 , 3.9 ⁇ M FeSO 4 , lignin depolymerization product 1-30 g/ L.
  • the fermented liquid is collected by centrifugation or ceramic membrane filtration, and the supernatant is placed in a storage tank for later use.
  • the collected bacterial cells are broken by a high pressure homogenizer, and then the supernatant is collected by filtration through a ceramic membrane, and then combined with the supernatant of the fermentation broth and placed in a storage tank.
  • the supernatant is passed through a polysulfone ultrafiltration membrane to remove other polymers such as impurity proteins, polypeptides, and polysaccharides, and the filtrate is collected.
  • Vanillin is prepared from the filtrate by conventional steps such as organic solvent extraction, concentration and crystallization.
  • the present invention relates to a novel method for synthesizing vanillin from various lignins by means of efficient microbial transformation. Due to its complex composition, lignin produces dozens of compounds after depolymerization, which are difficult to separate, purify and utilize.
  • the main product vanillin can be obtained by transforming the lignin and the depolymerized products through the biosynthesis of genetically engineered microorganisms. It provides an effective method for the high-value utilization of lignin.
  • microbial synthesis of vanillin is considered to be a kind of natural vanillin, which can be used as an additive for natural flavors.
  • Fig. 1 is a schematic diagram of vanillin dehydrogenase gene knockout
  • Figure 2 shows the content of vanillin in the fermentation broth detected by HPLC.
  • the present invention is a method for synthesizing vanillin through microbial transformation using lignin as a substrate.
  • the bacteria depolymerize and transform to synthesize vanillin and extract it into the organic solvent phase, and then further refine and purify.
  • the lignin biomass as a substrate is not particularly limited as long as it is a plant-derived biomass containing lignin containing phenylpropane units as one of the main components.
  • Specific examples thereof include poplar, willow, pine, sawdust, miscanthus, switchgrass, sorghum straw, corn straw, rice straw, wheat straw, bagasse, rice husk meal, wheat bran, alkaline lignin, lignosulfonic acid Sodium, sodium lignin sulfate, ground wood lignin, xylose residue, vinegar grains, papermaking black liquor, etc.
  • the microorganism selected in the present invention is a typical strain of Bacillus ligninophilus L1.
  • the vanillin dehydrogenase gene knockout method of Bacillus ligninophilus L1 selected in the present invention may be a homologous arm recombination method or a CRISP-Cas9 gene editing method or other methods capable of inactivating the vanillin dehydrogenase gene.
  • the lignin depolymerization methods include but are not limited to thermal cracking, enzymatic methods, microwaves, metal catalysts, and alkali catalysis methods.
  • Depolymerization in the present invention means that the lignin structure is capable of producing monomeric aromatic compounds such as ferulic acid and p-coumaric acid.
  • the culture medium and culture method used for culturing the genetically engineered bacteria are not particularly limited, and suitable nutrient components and culture methods for the growth and transformation of the bacteria can be appropriately selected.
  • the cultivation time is also not particularly limited, as long as the lignin depolymerization product can be converted to synthesize the desired target amount of vanillin.
  • the preferred bacterial growth medium is 2216E medium (component g/L, peptone 5, yeast powder 1, ferric citrate 0.1, sodium chloride 19.45, magnesium chloride 5.97, sodium sulfate 3.24, calcium chloride 1.8, sodium carbonate 0.16, bromine Potassium chloride 0.08, strontium chloride 0.034, boric acid 0.022, sodium silicate 0.004, sodium fluoride 0.0024, sodium nitrate 0.0016, sodium dihydrogen phosphate 0.008).
  • 2216E medium component g/L, peptone 5, yeast powder 1, ferric citrate 0.1, sodium chloride 19.45, magnesium chloride 5.97, sodium sulfate 3.24, calcium chloride 1.8, sodium carbonate 0.16, bromine Potassium chloride 0.08, strontium chloride 0.034, boric acid 0.022, sodium silicate 0.004, sodium fluoride 0.0024, sodium nitrate 0.0016, sodium dihydrogen phosphate 0.008).
  • the culturing time of the genetically engineered bacteria is not particularly limited, as long as the lignin depolymerization product can be converted to synthesize the desired target amount of vanillin, preferably 24 hours.
  • the yield of vanillin in the above case is not particularly limited depending on the purpose, and the content of vanillin in the unit medium can be, for example, 0.2 mg/L or more, preferably 100 mg/L or more, and more preferably 300 mg/L or more. .
  • vanillin synthesis conversion method of the present invention when extracting vanillin from the above-mentioned culture solution, it may be directly extracted from the microbial culture solution, or if necessary, the above-mentioned bacterial cells may be disrupted into a cell disrupted product and extracted from the disrupted product. Extraction and crushing can also be performed simultaneously.
  • the organic solvents used in the vanillin extraction in the present invention include but are not limited to methanol, ethanol, acetone, butanone, cyclohexanone, diethyl ether, petroleum ether, n-hexane, ethyl acetate, butyl acetate, n-propyl acetate, dimethyl acetate
  • One or more of base sulfoxide and the like are mixed in different ratios.
  • the bacterial cell separation method can be either a centrifugation method or a ceramic membrane filtration method.
  • the filtrate passes through the ultrafiltration membrane to further remove biological macromolecules such as polysaccharides, proteins, and cell wall fragments.
  • the purification method can be ion exchange column, macroporous resin or silica gel column elution.
  • Example 1 Construction of L1-vdh gene knockout engineering bacteria by homology arm method
  • Use primer design software (Primer Premier 5.0) software to design upstream and downstream homology arm primers within 500bp of the vanillin dehydrogenase gene vdh sequence (SEQ ID NO.1), and add restriction sites (KpnI, HindIII, NotI and PstI) (see Table 1 for the primers designed to construct gene knockout bacteria).
  • Construction of cloning vector The upstream and downstream homology arms of the target gene (named ch1-ch7) were cloned by PCR, and the product was purified and connected to pMD19-T plasmid and transformed into E.
  • knockout vector digest the upstream positive cloning plasmid and pKS1 thermosensitive plasmid with endonucleases KpnI and HindIII, respectively, to obtain a homologous fragment carrying the same cohesive end, clone the upstream homologous fragment into pKS1 and double-enzyme digestion to verify the positive Cloning (pKS1-ch-up).
  • the pT-ch-down and pKS1-ch-up were digested with endonucleases NotI and PstI, respectively, and the double-enzyme digestion products were recovered and purified.
  • the downstream homologous fragment was cloned into the pKS1-ch-up plasmid to verify the positive clone (pKS1-ch- up-down).
  • the positive clone pKS1-ch-up-down was transferred to the competent cells of bacterial L1 by electroporation, and the positive bacteria were obtained by the temperature-induced knockout method.
  • the engineering bacteria named Bacillus ligniniphilus L1-vdh, has been deposited in the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.
  • the lignin mixture was then hydrothermally depolymerized in a batch autoclave at 280°C for 60 minutes, and the pressure was 0.5- 6Mpa.
  • the depolymerized product is placed in a storage tank for later use.
  • Example 3 Fermentative transformation of alkali lignin by engineering bacteria
  • the peak area reference standard curve detection and calculation showed that the yield of vanillin in 24-hour shake flask culture had reached the highest value of 123mg/L, while the L1 wild strain (preserved in the laboratory) was only 6.4mg/L.
  • the yield of vanillin of engineering bacteria is 19.2 times that of wild bacteria (wild strain is inoculated in 2216E medium test tube and expanded for 24h, inoculated into a 500ml conical flask containing 100ml MM63 medium (containing alkaline lignin 10g/L), other fermentation conditions and
  • the detection methods are the same as those of genetically engineered bacteria)
  • Example 4 Fermentative transformation of lignin depolymerization mixture by engineered bacteria
  • the fermentation broth was filtered through a ceramic membrane, and the supernatant was collected.
  • the filtration speed of the ceramic membrane was 8 m/h, the pressure range was 0.4-1 Mpa, and the temperature was 40°C.
  • the supernatant was placed in a storage tank for later use.
  • the cell pellet was broken by a homogenizer and then centrifuged at a high speed of 16,000 rpm, and the supernatant was collected and combined into two supernatants. Then the supernatant is subjected to ultrafiltration through a 10-100 nm polysulfone membrane with a molecular weight cut-off of more than 2500, and the filtrate is collected.
  • the filtrate was continuously extracted with petroleum ether at a ratio of 1:1 (v/v), and then concentrated under reduced pressure through a two-effect evaporator to recover the petroleum ether to obtain a saturated concentrated solution.
  • the saturated concentrated liquid is pumped into the first-stage crystallizing tank, and then the saturated feed liquid enters the second-stage crystallizing tank from the first-level crystallizing tank. Then enter the third stage crystallizing tank. Finally, the crystallization rate is above 80%.
  • the crystallized vanillin was washed with cold water and dried to obtain the finished product, and the purity detected by HPLC was 99.5%.
  • Embodiment 6 Utilize papermaking black liquor to be the substrate transformation synthesis vanillin and extraction and separation
  • the papermaking black liquor is spray-dried, and the dry powder of the papermaking black liquor is collected for use.
  • the genetically engineered bacteria L1-vdh was inoculated into the 2216E medium (containing kanamycin 10 ⁇ g/L), the flask was expanded for 24 hours, and then inoculated into a 50L fermentor, the fermentation medium was 2216E medium, and the fermentation was replaced after 18 hours.
  • the content of vanillin in the culture medium was detected by HPLC to be 1.23 g/L.
  • the fermentation broth is centrifuged at 5000 rpm after the homogenizer is broken, and the supernatant is collected.
  • the supernatant is extracted with 5 times the volume of ether, and then concentrated under reduced pressure to recover the ether to obtain a vanillin concentrate.
  • the vanillin concentrate, the vanillin concentrate and 10 times the weight of styrene non-polar macroporous resin are stirred evenly and packed into a column for adsorption, and the pH is adjusted to 6-7.
  • the purity by HPLC was 98%. The yield was 7.6%.

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Abstract

L'invention concerne une bactérie génétiquement modifiée pour transformer une biomasse contenant de la lignine pour synthétiser de la vanilline et son utilisation. La bactérie a été nommée Bacillus ligniniphilus L1-vdh, et déposée dans le "Centre chinois pour la collection de souches cellulaires" en janvier 2020 avec un numéro d'accession CGMCC No. 19226. En neutralisant le gène codant pour la vanillate déshydrogénase de Bacillus ligniniphilus L1, le gène peut être inactivé, accumulant une grande quantité de vanilline et réalisant la transformation de la lignine en vanilline.
PCT/CN2020/126908 2020-09-23 2020-11-06 Bactérie génétiquement modifiée pour transformer une biomasse contenant de la lignine pour synthétiser de la vanilline et son utilisation WO2022062088A1 (fr)

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