Production strain for synthesizing acetoin by using xylose and application of production strain
By enhancing the specific protein function in salt monocylla, the use of cellulose hydrolyzed solution as a substrate realizes the efficient production of salt monocycus synthesis B marriage, solving the problem of low efficiency in existing technologies, and is a couple marriage. The industrialized production provides a new way.
Patent Information
- Application Number
- CN202411628304.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-01-28
AI Technical Summary
In the prior art, few studies have been conducted to synthesize Ethionin using cellulose hydrolysate fermented by salmonas, and the efficiency of cellulose hydrolysate being absorbed by salmonas is low, making it difficult to achieve efficient production.
By enhancing the specific protein function in salt monocylla, the use of cellulose hydrolyzed liquid as a substrate to catalyze the Bya marriage by whole cells. Specific methods include expressing the protein function of XYLA, Xylb, ALSD, and ALSS genes. The protein function is enhanced through the MMP1-IPTG induction system and PPORIN component system.
The efficient production of Salithic Monosenococcus with Crystone Hydrolyzed Salt Sumidia is achieved, which provides a new way for the industrialized production of Bya's marriage.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to a production strain that utilizes xylose to synthesize acetoin and an application thereof. Background Art
[0002] Biomass is widely available and abundant, and is a promising green and economic raw material for the production of chemicals. The main available components of cellulosic biomass after hydrolysis are glucose and xylose, of which xylose accounts for 30% of the total sugar content in cellulose hydrolysate and can be used as a source for the production of various basic chemicals. Therefore, the effective utilization of xylose is a necessary condition for the development of lignocellulosic biomass. Acetoin, with the chemical formula C4H8O2, is easily soluble in water, soluble in ethanol and propylene glycol, slightly soluble in ether, and almost insoluble in vegetable oil. It can be used as a edible flavor in the preparation of cream, dairy products, yogurt, etc., and can also be used in organic synthesis. It is an important platform compound. As a fermentation bacterium, Halomonas can be open fermented without additional sterilization. It is a fermentation microorganism with great application potential. If Halomonas can be used to ferment cellulose hydrolysate to produce acetoin, it will not only realize the resource development and effective utilization of lignocellulosic biomass, but also realize the safe, environmentally friendly and efficient production of acetoin. However, due to the microorganisms' preference for nutrient sources, the efficiency of cellulose hydrolysate absorption by Halomonas is low, and there are few studies on the synthesis of acetoin using Halomonas. So far, there has been no report on the synthesis of acetoin by fermenting cellulose hydrolysate with Halomonas.
[0003] In view of this, the present invention is proposed. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a production strain for producing acetoin based on cellulose hydrolyzate and whole-cell catalysis of Halomonas and its application, which provides a new idea for the production of acetoin.
[0005] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a production strain that utilizes xylose to synthesize acetoin, wherein the production strain uses cellulose hydrolyzate as a substrate; the production strain is a Halomonas strain that enhances at least one of the following protein functions ① and at least one of the following protein functions ②; ①: XYLA, XYLB; ②: ALS, ALDC.
[0006] Preferably, the method for enhancing protein function is selected from at least one of the following: overexpressing a gene corresponding to the protein, and enhancing the biological activity of the protein.
[0007] Preferably, the Halomonas overexpresses the following genes by mmpl-IPTG induction system: xylA, xylB ; Preferably, the plasmid structure of the mmp1-IPTG induction system is: pSEVA321-mmp1 -xylA - xylB .
[0008] Preferably, the Halomonas overexpresses the following genes via the Pporin constitutive system: alsd and alss ; Preferably, the plasmid structure of the Pporin constitutive system is: pSEVA341-Pporin- alsd - alss .
[0009] Preferably, the amino acid sequence of XYLA is shown as SEQ ID NO.01; and / or, the amino acid sequence of XYLB is shown as SEQ ID NO.02; and / or, the amino acid sequence of ALS is shown as SEQ ID NO.03; and / or, the amino acid sequence of ALDC is shown as SEQ ID NO.04.
[0010] Preferably, xylA The nucleotide sequence is shown in SEQ ID NO.05; and / or, xylB The nucleotide sequence is shown in SEQ ID NO.06; and / or, alsd The nucleotide sequence is shown in SEQ ID NO.7; and / or, alss The nucleotide sequence is shown in SEQ ID NO.8.
[0011] Preferably, the induction concentration of IPTG is 1-100 mg / L, more preferably 10±5 mg / L.
[0012] In a second aspect, the present invention provides use of the production strain in preparing acetoin or downstream products containing acetoin.
[0013] In a second aspect, the present invention provides a method for preparing acetoin, which utilizes the production strain and cellulose hydrolyzate as a substrate to produce acetoin through whole-cell catalysis.
[0014] Preferably, the conditions for whole-cell catalysis include: 37±2°C, 200±50 rpm, pH 7.5-8.5, and reaction time of 48±6 h.
[0015] In the more specific experimental scheme provided by the present invention, the most suitable content of cellulose hydrolyzate (derived from Novizan) is selected, and the concentration is 80%. Comparing 2mL 4mL 6mL 8mL and 10mL cellulose hydrolyzate and 30g / L glucose, the content of acetoin that can be synthesized is 0.1 0.3 0.4 0.55 and 0.59 g / L respectively. Therefore, the content of cellulose hydrolyzate in the present invention is preferably 10 mL.
[0016] The preparation method provided by the invention has simple production process, low cost and broad application prospect.
[0017] Beneficial effects: The present invention provides a production strain for synthesizing acetoin using xylose and its application. The production strain of the present invention uses cellulose hydrolyzate as a substrate, specifically a Halomonas strain that enhances the function of at least one protein in XYLA and XYLB and enhances the function of at least one protein in ALS and ALDC. The present invention enhances the specific protein function of Halomonas strain by biological means, realizes the Halomonas strain synthesis of acetoin using cellulose hydrolyzate as a substrate, and provides a new approach for the industrial production of acetoin. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.
[0019] Figure 1 Schematic diagram of the metabolic pathway for synthesizing acetoin from xylose in the present invention.
[0020] Figure 2 Schematic diagram of the structure of plasmid 1 in the embodiment of the present invention.
[0021] Figure 3 Schematic diagram of the structure of plasmid 2 in an embodiment of the present invention. DETAILED DESCRIPTION
[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.
[0023] In the present invention, the proteins involved, their corresponding genes and their corresponding full names are as follows: XYLA, xylA , xylose isomerase; XYLB, xylB , xylulose kinase; ALS, alsd ,α-acetolactate synthetase; ALDC, alss , α-acetolactate decarboxylase.
[0024] The specific sequence information is as follows: SEQ ID NO.01(XYLA): MNYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVESVRRLAELGAHGVTFHDDDLIPFGSSDSEREEHVKRFRQALDDTGMKVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTIRNIDLAVELGAETYVAWGGREGAESGGAKDVRDALDRMKEAFDLLGEYVTSQGYDIRFAIEPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGKLFHIDLNGQNGIKYDQDLRFGAGDLRAAFWLVDLLESAGYSGPRHFDFKPPRTEDFDGVWASAAGCMRNYLILKERAAAFRADPEVQEALRASRLDELARPTAADGLQALLDDRSAFEEFDVDAAAARGMAFERLDQLAMDHLLGARG。
[0025] SEQ ID NO.02(XYLB): MKYVIGIDLGTSAVKTILVNQNGKVCAETSKRYPLIQEKAGYSEQNPEDWVQQTIEALAELVSISNVQAKDIDGISYSGQMHGLVLLDQDRQVLRNAILWNDTRTTPQCIRMTEKFGDHLLDITKNRVLEGFTLPKMLWVKEHEPELFKKTAVFLLPKDYVRFRMTGVIHTEYSDAAGTLLLHITRKEWSNDICNQIGISADICPPLVESHDCVGSLLPHVAAKTGLLEKTKVYAGGADNACGAIGAGILSSGKTLCSIGTSGVILSYEEEKERDFKGKVHFFNHGKKDSFYTMGVTLAAGYSLDWFKRTFAPNESFEQLLQGVEAIPIGANGLLYTPYLVGERTPHADSSIRGSLIGMDGAHNRKHFLRAIMEGITFSLHESIELFREAGKSVHTVVSIGGGAKNDTWLQMQADIFNTRVIKLENEQGPAMGAAMLAAFGSGWFESLEECAEQFIREAAAFYPKAQNVQKYKTLFDLYKNIYTHTKDLNTALKSFRKN。
[0026] SEQ ID NO.03 (ALS): MKRESNIQVLSRGQKDQPVSQIYQVSTMTSLLDGVYDGDFELSEIPKYGDFGIGTFNNKLDGELIGFDGEFYRLRSDGTATPVQNGDRSPFCSFTFFTDMTHKIDAKMTREDFEKEINSMLPSRNLFYAIRIDGLFKKVQTRTVELQEKPYVPMVEAVKTQPIFNFDNNVRGTIVGFLTPAYANGIAVSGYHLHFIDEGRNSGGHVFDYVLEDCTVTISQKMNMNLRPNTADFFNANLDNPDFAKDIETTEGSPE.
[0027] SEQ ID NO.04(ALDC): 。
[0028] SEQ ID NO.05( xylA ):
[0029] SEQ ID NO.06( xylB ):
[0030] SEQ ID NO.07( alsd ): ATGAAACGAGAAAGCAACATTCAAGTGCTCAGACGCGGTCAAAAAGATCAGCCTGTGAGCCAGATTTATCAAGTATCAACAATGACTTCTCTATTAGACGGAGTATATGACGGAGATTTTGAACTGTCAGAGATTCCGAAATATGGAGACTTCGGTATCGGAACCTTTAACAAGCTTGACGGAGAGCTGATTGGGTTTGACGGCGAATTTTACCGTCTCCGTTCAGACGGAACCGCGACACCGGTCCAAAACGGGGATCGTTCTCCGTTCTGTTCATTTACGTTCTTTACACCGGACATGACGCACAAAATTGATGCGAAAATGACACGCGAAGACTTTGAAAAAGAGATCAACAGCATGCTGCCAAGCAGAAACTTATTTTATGCAATTCGTATTGACGGATTGTTTAAAAAGGTGCAGACAAGAACGGTAGAACTTCAAGAAAAACCTTACGTGCCAATGGTTGAAGCGGTCAAAACACAGCCGATTTTCAACTTCGACAACGTGAGAGGAACAATCGTAGGTTTCTTGACACCAGCTTATGCAAACGGAATCGCCGTTTCAGGCTATCACCTGCACTTCATTGACGAAGGACGCAATTCAGGCGGACACGTTTTTGACTATGTGCTTGAGGATTGCACGGTTACGATTTCTCAAAAAATGAACATGAATCTCAGACTTCCGAACACAGCGGATTTCTTTAATGCGAATCTGGATAACCCTGATTTTGCGAAAGATATCGAAACAACTGAAGGAAGCCCTGAATAA。
[0031] SEQ ID NO.08( alss ):
[0032] Example 1 (A) The construction process of Halomonas.
[0033] Construct a dual-plasmid system: Plasmid 1: pSEVA321-mmp1-xylA-xlyB (optimum IPTG is 10 mg / L); Plasmid 2: pSEVA341-Pporin58-alsd-alss.
[0034] (ii) The experimental process, results and conclusions of using the constructed Halomonas to ferment cellulose hydrolysate to synthesize acetoin.
[0035] Halomonas overexpressed plasmid 1 and plasmid 2, and the shake flask fermentation conditions were 60 LB. The specific scheme is shown in Table 1 below.
[0036] Table 1
[0037] The specific culture conditions of LB60 are 5 g / L yeast extract, 10 g / L peptone, and 60 g / L NaCl. The control group is 60LB medium, while the experimental group is 60LB medium with 10 mL of cellulose hydrolyzate added initially, 37°C, 200 rpm, cultured for 48 h, IPTG is 10 mg / L, the control group does not synthesize acetoin, while the experimental group can synthesize 0.51 g / L.
[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A production strain for synthesizing acetoin using xylose, characterized in that: The production strain uses cellulose hydrolyzate as a substrate; the production strain is a Halomonas strain that enhances at least one protein function as follows ① and at least one protein function as follows ②; ①: XYLA, XYLB; ②: ALS, ALDC.
2. The production strain for synthesizing acetoin using xylose according to claim 1, characterized in that: The method for enhancing the function of the protein is selected from at least one of the following: overexpressing the gene corresponding to the protein, and enhancing the biological activity of the protein.
3. The production strain for synthesizing acetoin using xylose according to claim 1, characterized in that: The Halomonas overexpressed the following genes through the mmp1-IPTG induction system: xyA , xy1 ; Preferably, the plasmid structure of the mmp1-IPTG induction system is: pSEVA321-mmp1- xylA-xylB .
4. The production strain for synthesizing acetoin using xylose according to claim 1, characterized in that: The Halomonas overexpresses the following genes via the Pporin constitutive system: alsd and alss ; Preferably, the plasmid structure of the Pporin constitutive system is: pSEVA341-Pporin -alsd-alss .
5. The production strain for synthesizing acetoin using xylose according to any one of claims 1 to 4, characterized in that: The amino acid sequence of XYLA is shown in SEQ ID NO.01; and / or, the amino acid sequence of XYLB is shown in SEQ ID NO.02; and / or, the amino acid sequence of ALS is shown in SEQ ID NO.03; and / or, the amino acid sequence of ALDC is shown in SEQ ID NO.
04.
6. The production strain for synthesizing acetoin using xylose according to claim 5, characterized in that: xyA The nucleotide sequence is shown in SEQ ID NO.05; and / or, xyB The nucleotide sequence is shown in SEQ ID NO.06; and / or, alsd The nucleotide sequence is shown in SEQ ID NO.7; and / or , alss The nucleotide sequence is shown in SEQ ID NO.
8.
7. The production strain for synthesizing acetoin using xylose according to claim 6, characterized in that: The induction concentration of IPTG is 1-100 mg / L, preferably 10±5 mg / L.
8. Use of the production strain according to any one of claims 1 to 7 in the preparation of acetoin or acetoin-containing downstream products.
9. A method for preparing acetoin, characterized in that: The production strain according to claims 1 to 7 is used to produce acetoin by whole-cell catalysis using cellulose hydrolyzate as a substrate.
10. The preparation method according to claim 9, characterized in that: The conditions for whole-cell catalysis include: 37±2°C, 200±50 rpm, pH 7.5-8.5, and reaction time of 48±6 h.