TWI652342B - Microrganism for forming fermentation products through fermentation of sugars - Google Patents

Abstract

The present invention relates to a microorganism for forming a fermentation product for fermentation by a fermentation, comprising a plastid comprising a mutated enzyme sequence selected from the group consisting of ribulose bisphosphate carboxylase/oxygenase and Related enzymes. Among them, the main object of the present invention is to fix carbon dioxide, thereby reducing carbon dioxide, and in the fermentation product of the gas phase, the proportion of hydrogen can be greatly increased due to the circulation of carbon dioxide. Therefore, the cost of purifying hydrogen can be further reduced.

Description

Microorganism for fermenting sugar to form a fermentation product

The present invention relates to a microorganism for fermentation, and more particularly to a microorganism capable of transforming a plastid containing a sequence of a mutated enzyme into a microorganism, thereby recovering carbon dioxide and improving fermentation efficiency.

All along, the production of fermented foods relies on microorganisms. Microorganisms are connected to raw materials. During the growth process, the required enzymes are produced, and the process of decomposing organic matter into beneficial substances in human life is called fermentation; therefore, this metabolic activity is actually catalyzed. The performer is an enzyme produced by microorganisms.

In the process of microbial fermentation, it can be divided into aerobic fermentation and anaerobic fermentation depending on whether oxygen is required. For example, mold culture and acetic acid fermentation are aerobic fermentation; alcohol fermentation and lactic acid fermentation are anaerobic fermentation. According to different raw material types and devices, it can be divided into solid fermentation and liquid fermentation, solid fermentation such as rice and sorghum fermentation. Because of the low water content of raw materials, the tolerance of mixed bacteria is high, and it is suitable for the cultivation of open space. For example, mold culture; liquid fermentation, because of high water content, rapid growth of microorganisms, easy to contaminate, it is often used in closed culture, such as the fermentation process requires oxygen, and then into the sterile air.

In recent years, in order to utilize the principle of microbial fermentation to prepare fermented products for daily life, there has been an industrial development of fermentation engineering. The fermentation process is used to solve the fermentation process. The field of engineering problems for industrial production. Fermentation engineering divides the fermentation industrial process that realizes the fermentation process into three stages: strain, fermentation and refining (including wastewater treatment) from the engineering point of view. These three stages have their own engineering problems, which are generally called respectively. It is the upstream, midstream and downstream projects of the fermentation project. The three stages of the fermentation project have their respective process principles and equipment and process control principles, which together constitute the principle of fermentation engineering.

Among them, fermentation engineering involves the fixation of carbon, and carbon enters the metabolic process of fermentation engineering in the form of carbon dioxide. The ribulose bisphosphate carboxylase/oxygenase (RubisCO) acts as an enzyme for converting carbon dioxide into biomass (mass) during the fermentation process. This is a common enzyme; however, ribulose bisphosphate carboxylase/oxygenase is currently a slower metabolic enzyme (Kcat~3/s) in bioengineering. It is not clear why its rate is so slow. And the specificity is low, and many of the general knowledge in the field are currently focused on using reasonable experimental design to enhance or improve fermentation efficiency.

However, in this fermentation process, the use of microorganisms to produce small molecule chemicals such as bioethanol or bio-butanol is focused on how to effectively produce it, and after taking into account the production of small molecule chemicals, once mass production is started, The result is a large amount of carbon dioxide emissions, such as the production of a molar ethanol will produce a mole of carbon dioxide, few people focus on the fixation or recycling of carbon dioxide and its further use. Only in U.S. Patent No. 20080085341, which provides a method for forming a fermentation product using at least one heterologous gene sequence which exhibits an exogenous protein capable of converting a foreign carbon source into and fixing carbon dioxide. It is a desirable fermentation product; although it has been mentioned that immobilized carbon dioxide does not increase the amount of carbon dioxide in the environment, it does not further improve industrial applications such as fermentation efficiency, productivity, and bioengineering.

In view of the above, it is an object of the present invention to provide a microorganism which can focus on the fixation and recovery of carbon dioxide during the fermentation process, thereby reducing the amount of carbon dioxide emissions in the fermentation industry and reducing the burden of excessive carbon dioxide on the earth.

At the same time, another object of the present invention is to transform a plastid containing a sequence of a mutant enzyme into a microorganism to enhance the efficiency of fermentation under the premise of fixing carbon dioxide.

In order to achieve the above object, the present invention provides a microorganism for forming a fermentation product by fermentation, which comprises a plastid or a chromosome containing a mutated enzyme sequence, and the mutated enzyme comprises a plastid selected from the following sequence structure or a group consisting of a chromosome: pET-rbcLS (J2), pET-rbcLSm1 (J3), pACYC184-PrkA/pET-rbcLS (JA), and pACYC184-PrkA/pET-rbcLSm1 (JB); The dry weight of cells after 25 hours of microbial fermentation of plastids or chromosomes is increased by 200%-300% compared with wild type strains.

In a preferred embodiment of the invention, the microorganism can be a fungus.

Preferably, the fungus can be a genus of yeast.

In another preferred embodiment of the invention, the microorganism can be a bacterium or an archaea or a combination thereof.

Preferably, the bacteria may be Escherichia coli, Clostridium acetobutylicum, Bacillus subtilis, Pseudomonas putia, cyanobacteria, greedy Cucurbitus (Cupriavidus spp.), or Zymomonas mobilis or a combination thereof.

In another preferred embodiment of the invention, the microorganism can be an alga.

Preferably, the substrate used in the fermentation process of the present invention may comprise a five carbon sugar, a six carbon sugar, a glycerin, an organic fatty acid or a combination thereof.

Wherein, the above five carbon sugars may comprise arabinose, xylose or a combination thereof.

Wherein, the above hexose carbon sugar may comprise glucose, galactose, fructose, mannose, sucrose, lactose or a combination thereof.

Many of the features and advantages of the present invention will become more apparent from the detailed description of the appended claims.

The first plot shows the growth curves of various strains only under aerobic conditions in LB medium.

The second graph shows the (A) growth curve and (B) sugar content of various strains in aerobic conditions of LB medium containing arabinose.

The third figure is a schematic diagram showing the ratio of carbon dioxide produced by fermentation of Escherichia coli to acetic acid, carbon dioxide and ethanol.

The detailed description of the present invention is intended to be illustrative of the embodiments of the invention, and is not intended to represent the invention. The order of the functions and steps mentioned in the embodiments is used to construct and operate the embodiments. However, the same or equivalent functions or sequences may be achieved by different embodiments.

For the sake of convenience, the embodiments employed in the specification, examples, and appended claims are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

The singular forms "a", "the", and "the"

The term "fixed" as used herein refers to the conversion of carbon dioxide and/or the incorporation of carbon atoms of carbon dioxide into organic molecules such that the carbon dioxide produced in the fermentation process can be recycled indefinitely.

The present invention provides a microorganism for forming a fermentation product by fermentation, which comprises a plastid or a chromosome containing a mutated enzyme sequence, which may be a nucleotide sequence or a protein sequence, and the mutated enzyme is selected from The group consisting of the following EC numbers (Enzyme Commission number): 2.7.1, 5.1.3, 5.3.1, 4.1.1, 2.2.1, and 3.1.3.

Among them, EC number 2.7.1 is preferably phosphoribulokinase (EC number: 2.7.1.19); EC number 5.1.3 is preferably ribulose-phosphate 3-epimerase (Ribulose-phosphate 3-epimerase, EC number: 5.1.3.1), L-ribulose-5-phosphate 4-epimerase (EC number: 5.1.3.4), 5-phosphate ribulose 5-phosphate 3-episoose-5-phosphate 3-epimerase (EC number: 5.1.3.22); EC number 5.3.1 is preferably 5-phosphate ribose isomerase / pentose phosphate isomerase ( Ribose 5-phosphate isomerase/phosphopentose epimerase, EC number: 5.3.1.6); EC number 4.1.1 is preferably ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCo ), EC number: 4.1.1.39); EC number 2.2.1 is preferably transaldolase (EC number: 2.2.1.2), acetoin-5-phosphatidulose aldolase (acetoin- Ribose-5-phosphate transaldolase, EC number: 2.2.1.4), fluorothreonine transaldolase (EC number: 2.2.1.8), transketolase (EC number: 2.2.1.1) A Transketolase (EC number: 2.2.1.3); EC number 3.1.3 is preferably sedoheptulose 1,7-bisphosphatase, EC number: 3.1. 3.37).

The fermentation product may be hydrogen, nucleotides, amino acids, alcohols such as ethanol, butanol, isobutanol, propylene glycol isomers, sorbitol, alkenes such as ethylene, propylene, isoprene. Isoprene, organic acids such as fatty acids, succinic acid, shikimic acid, acetic acid, esters such as biodiesel, Poly(3-hydroxybutyrate), PHB), alkylene hydrocarbons, organic acids such as succinic acid, itaconic acid, acetamidine Levulinic acid, 3-hydroxypropionic acid, polylactic acid, and biomass.

Among them, poly(3-hydroxybutyrate) is a biodegradable material

Among them, the microorganism of the present invention can be used for fixing carbon dioxide.

In a preferred embodiment of the invention, the microorganism can be a fungus (Fungi).

Among them, the above fungus may be a genus of the genus Saccharomycesspp.

In another preferred embodiment of the invention, the microorganism can be a bacterium.

Among them, the above bacteria may be Escherichia coli, Clostridium acetobutylicum, Bacillus subtilis, Pseudomonas putia, cyanobacteria, greedy Cucurbitus (Cupriavidus spp.) or Zymomonas mobilis or a combination thereof.

In another preferred embodiment of the invention, the microorganism can be an algae.

Preferably, the sugar used in the fermentation process of the present invention may comprise a five carbon sugar or a six carbon sugar.

Wherein, the above five carbon sugars may comprise arabinose, xylose or a combination thereof.

Wherein, the above hexose carbon sugar may comprise glucose, galactose, fructose, mannose, sucrose or a combination thereof.

In addition, the present invention can continuously fix the carbon dioxide generated after the fermentation, and the gas of the fermentation product only leaves hydrogen gas. Therefore, the present invention can also improve the efficiency of hydrogen removal and reduce the cost of hydrogen purification.

The examples described below are provided to illustrate certain aspects of the invention and to assist those skilled in the art to practice the invention. These examples are not to be construed as limiting the scope of the invention.

Instance

The present invention will further describe the nature and application of the modified microorganisms in the fermentation with reference to the following examples, which are intended to be illustrative only and not intended to limit the scope of the invention.

Example 1

The strain used in the fermentation process in this experiment is E.coli BL21(DE3) (hereinafter referred to as Escherichia coli), and the enzyme to be transferred into Escherichia coli is the mutated ribulose bisphosphate carboxylase/oxygenase. (RbcLS) and mutated ribulose kinase (PrkA). The allele mutation sequence of the two enzymes is referred to Parikh et. al., Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli, 2006, Protein Engineering, Design & Selection, 19:113. In the method of treatment, the mutated sequence itself, its N-terminal coding, restriction enzymes and the like, and the subclone method, and the treatment method of the strain of the present invention are the same as the paper, which is a common knowledge in the field. The known techniques are not described here. After treatment with each step, the final structure of the treated plasmid was pACYC184-PrkA, pET-rbcLS, pET-rbcLSm1, and transformed into E. coli and named J1, J2 and J3, respectively. In addition, pACYC184-PrkA and pET-rbcLS were additionally transformed into Escherichia coli, which was referred to as JA; pACYC184-PrkA and pET-rbcLSm1 were also co-transformed into Escherichia coli, which was referred to as JB. In addition, although the present embodiment inserts the condition of the desired sequence into the plastid, it is merely exemplary and not limiting. For example, the present invention can also insert the condition of the desired sequence into the chromosome and insert the chromosome. The advantage is that the bioengineering system of the invention presents a more stable state. Furthermore, this embodiment uses the T7 startup subsystem for testing, but it is not limiting, and any conventional startup subsystem can be applied to the present invention. It should be noted that this example only uses the above-mentioned mutant enzyme as an experimental description, and other related mutant-treated enzymes, such as: ribulose bisphosphate carboxylase activating enzyme, phosphoribril kinase, 1 , 5-ribulose ribulose epimerase, ribulose bisphosphate carboxylase/oxygenase type I, ribulose bisphosphate carboxylase/oxygenase type II, ribulose diphosphate Carboxylase/oxygenase type III, ribulose bisphosphate carboxylase/oxygenase type IV, Or enzymes that metabolize sugar-related substances, such as: pentose phosphate isomerase, 5-phosphate ribose isomerase, transaldolase, transketolase, and sedoheptulose-1,7-diphosphate Enzymes, etc., can be used in this experiment.

Example 2

The culture medium used for the activation of the strain is a sterile protein medium (Luria Broth Medium) (hereinafter referred to as LB medium), and the composition thereof is NaCl 10 g/L; tryptone 10 g/L; and yeast extract. (Yeast Extract) 5g/L. Among them, the wild-type Escherichia coli was not added with antibiotics and was used as a control group; the antibiotic applied in J1 was chloramphenicol (Chloramphenicol) 34 μg; J2 and J3 were all administered with Kanamycin 50 μg; JB was applied with 34 μg of chloramphenicol and 50 μg of oxytetracycline. Isopropyl-β-D-thiogalactoside (IPTG) was selectively added during the cultivation according to the needs of the control group or the experimental group.

After that, the batch culture procedure was carried out, and Escherichia coli was cultured in a 125 mL sterile grooved conical flask. The Erlenmeyer flask contained 25 mL of LB medium, and the culture environment was activated in an aerobic environment at 37 ° C, 200 r. pm for 12 hours (preculture) ) to increase the activity of the cells and to take samples at appropriate times.

Next, the fermentation tank is subjected to semi-anaerobic fermentation in a 3L tank, and 1L LB medium is in the tank; the culture environment is 37 ° C, 150 r. pm, and the amount of carbon dioxide dissolved in the LB medium is continuously observed, and then taken at intervals. , metabolite analysis and sugar content in LB medium.

Wherein, the LB medium used contains 20 g/L of arabinose (L-Arabinose) and the corresponding antibiotic; however, the arabinose is merely exemplary in the embodiment, not limiting, and other five carbon sugars, For example, xylose or a combination with other five carbon sugars, as well as six carbon sugars such as glucose, galactose, fructose, mannose or combinations thereof, can be readily utilized in this experiment.

Example 3

After the above activation step, the cell density of Escherichia coli is measured by a spectrophotometer, and the amount of bacteria in the main culture is determined by the value of OD600. This step is mainly to measure the concentration of E. coli culture solution by absorbance of Escherichia coli, thereby estimating The growth of E. coli, so it is usually used to refer to Cell density. Before the measurement, warm up for 10 minutes, then adjust the wavelength to 600nm to measure the absorption value of each sample; firstly, put the blank sample into the zero adjustment operation, if the absorption value is greater than 0.8, dilute to the appropriate magnification, and then measure the absorption. Value; at the beginning of the culture, the initial OD600 value of each cell is about 0.050.

Example 4

A quantitative test of arabinose was carried out during fermentation to compare the fermentation efficiency. First, take a 1m sample and centrifuge at 13,300rpm for 10 minutes. Then remove the supernatant and dilute to the appropriate ratio. Add DNS reagent (10g/L dinitrosalicylic acid, 16g/) in a ratio of 1:1. L sodium hydroxide and 300 g/L potassium sodium tartrate). Then, the mixture was heated in a water bath at 100 ° C for 10 minutes in the dark, and then cooled in the dark for 5 minutes. Finally, the absorption value of the wavelength at 550 nm is measured by a spectrophotometer. If the absorption value is greater than 0.8, the dilution is performed to an appropriate magnification, and the absorption value is measured.

The content of reducing sugar in the fermentation broth can be known from the previous calibration curve.

Results and discussion

Please refer to the first figure. The first figure shows the growth curves of various strains only under aerobic conditions in LB medium. As can be seen from the first figure, the OD600 values of the strains were not significantly different within 10 hours from the beginning, and after 30 hours, the diploid Escherichia coli (JA, JB) showed a higher OD600 value than the wild type (WT). ) E. coli is 17.1% lower. In contrast, please refer to the second figure. The second figure shows the (A) growth curve and (B) sugar content of various strains in the aerobic condition of LB medium containing arabinose.

Comparing the first figure with the second (A) figure, it can be found that the OD600 value of each strain showed little difference in the first 10 hours, and the OD600 value showed a significant increase between 10 and 30 hours. (especially J2, J3, JA and JB strains), further, after 30 hours, or even 100 hours, it can be found that the OD600 value of J1 is similar to WT, and the other strains (J2, J3) The OD600 values of JA, JA and JB are much larger than those of WT. It can be clearly seen from the second (A) chart that the dry weight of cells of J2, J3, JA and JB strains increased by about 200%-300 compared with WT 25 hours after fermentation. %; it can be clearly inferred that the OD600 value of the E. coli strain with the mutated ribulose bisphosphate carboxylase/oxygenase (rbcLS) is significantly higher than that of the E. coli strain. Preferably, the transmutation of the ribulose bisphosphate carboxylase/oxygenase (rbcLS) into E. coli can make it grow more efficiently and significantly increase the dry weight of the cells and the metabolism of the carbon source.

At the same time, please also refer to the second (B) chart. The initial arabinose content is 20g/L. In 10 hours, the sugar consumption of each strain is similar; however, between 10 and 30 hours, sugar consumption Significantly increased the amount (especially J2, J3, JA and JB strains), after 30 hours, or even 100 hours, it can be found that the sugar consumption of J1 is similar to that of WT, and the other strains (J2) The sugar consumption of J3, JA and JB) is significantly greater than that of WT; it can be clearly inferred that the Escherichia coli strain with the mutated ribulose bisphosphate carboxylase/oxygenase (rbcLS) can increase the fermentation rate, The fermentation efficiency is increased and the sugar consumption is significantly increased.

Please refer to the third figure. The third figure is a schematic diagram showing the ratio of carbon dioxide produced by fermentation of Escherichia coli to acetic acid, carbon dioxide and ethanol. As shown, the ratio of carbon dioxide to acetic acid in wild-type E. coli is much higher than that of other plants, indicating that the carbon dioxide of wild-type plants does not have the ability to fix and recover carbon dioxide, thus producing a lot of carbon dioxide. At the same time, the ratio of carbon dioxide to ethanol in wild plants is also higher than that of JB, J1 and J2. It can be seen that the large amount of mutant enzymes in E. coli can help to fix and recover carbon dioxide and reduce carbon dioxide emissions.

In summary, the microorganism of the present invention for forming a fermentation product by fermentation is provided with the following advantages:

(1) Microorganisms containing a mutated enzyme sequence can increase the production efficiency of the fermentation product, and fix carbon dioxide to reduce carbon dioxide emissions.

(2) Increasing the proportion of hydrogen in the gas phase product can reduce the cost of hydrogen purification.

(3) A large number of recombinant protein enzymes can significantly increase cell dry weight and carbon source metabolism in an aerobic or anaerobic environment.

Other implementations

All features disclosed in this disclosure can be combined in any manner. Features disclosed in this specification can be replaced with features of the same, equivalent or similar purpose. Therefore, the features disclosed in this specification are one of a series of equivalent or similar features.

In addition, according to the disclosure of the present specification, those skilled in the art can easily make appropriate changes and modifications to different methods and situations without departing from the spirit and scope of the present invention. The implementation aspect is also included in the scope of the patent application.

Claims (10)

A use of a microorganism for fermenting and recovering carbon dioxide, the microorganism comprising a plastid or chromosome comprising a mutated enzyme sequence, the mutated enzyme comprising a plastid or chromosome selected from the following sequence structure a group consisting of: pET-rbcLS, pET-rbcLSm, pACYC184-PrkA/pET-rbcLS, and pACYC184-PrkA/pET-rbcLSm1; wherein the receptor is a five-carbon sugar, glycerin, an organic fatty acid or a combination thereof; The microorganisms recover carbon dioxide. The use of the microorganism according to item 1 of the patent application, wherein the microorganism is a fungus. The use of the microorganism according to item 2 of the patent application, wherein the fungus is a genus of yeast. The use of the microorganism according to item 1 of the patent application, wherein the microorganism is a bacterium or an archaea or a combination thereof. The use of the microorganism according to item 4 of the patent application, wherein the bacteria are Escherichia coli, Clostridium acetobutylicum, Bacillus subtilis, Pseudomonas Putia), cyanobacteria, Cupriavidus spp. or Zymomonas mobilis or a combination thereof. The use of the microorganism according to item 1 of the patent application, wherein the microorganism is an alga. The use of the microorganism according to item 1 of the patent application, wherein the five-carbon sugar comprises arabinose, xylose or a combination thereof. The use of the microorganism according to the first aspect of the patent application, wherein the microorganism is subjected to fermentation engineering in a half anaerobic environment. The use of the microorganism according to the first aspect of the patent application, wherein the microorganism is subjected to a fermentation process in a liquid fermentation tank. According to the use of the microorganism of claim 1, wherein the mutation positions of J2, J3, JA and JB are A8S/M259T, M259T or M259T/F342S of the rbcLS sequence.