Genetic engineering transformation method for improving generation of acetic acid and application of genetic engineering transformation method
By performing targeted genetic engineering on Escherichia coli, knocking out or mutating related genes, the problem of acetic acid production in high-density E. coli fermentation was solved, resulting in reduced acetic acid production and improved fermentation process, thus increasing production efficiency and consistency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-08
- Publication Date
- 2026-04-10
AI Technical Summary
Escherichia coli is prone to producing acetic acid during high-density fermentation, which leads to carbon source waste, inhibits cell growth and exogenous gene expression, increases the difficulty of process control and batch-to-batch inconsistency in the fermentation process, and existing strategies have limited universality and are time-consuming and labor-intensive.
By performing targeted genetic engineering on Escherichia coli, the genes galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl can be knocked out or mutated to reduce acetic acid production.
It significantly reduces the production of acetic acid byproducts, is simple and easy to implement, has a wide range of applications, improves fermentation processes, and increases production capacity and batch consistency.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial biotechnology and genetic engineering, specifically relating to a genetic engineering method for improving acetic acid production, a recombinant prokaryotic cell with improved acetic acid production, and its application. Background Technology
[0002] Escherichia coli (especially K12 series strains) readily produces acetic acid as a metabolic byproduct during high-density fermentation. Excessive acetic acid leads to the waste of a major carbon source, inhibits cell growth and the expression of exogenous genes, thereby severely limiting the production capacity of E. coli and increasing the difficulty of process control and batch-to-batch inconsistencies during fermentation.
[0003] Traditional strategies for addressing acetic acid production during E. coli fermentation mainly focus on the following aspects: (1) strengthening process control during fermentation, such as optimizing the culture medium, controlling the feeding rate, controlling DO (dissolved oxygen), and pH; (2) targeted modification strategies based on metabolic engineering, such as constructing mutant strains of the pyruvate oxidase (PoxB) pathway and the phosphoacetyltransferase / acetylkinase (Pta / AckA) pathway related to acetic acid production; and (3) breeding strategies, such as screening for acetic acid-tolerant strains or mutant strains that reduce acetic acid production. However, due to the differences between strains, the universality of the above strategies is limited, and developing customized strategies for different strains is time-consuming, labor-intensive, and increases additional costs and time.
[0004] Therefore, there is still an urgent need in this field for a simple, easy-to-implement, and effective universal strategy to improve the problem of acetic acid production. Summary of the Invention
[0005] To address the aforementioned technical problems, the inventors have developed a genetic engineering strategy that can significantly reduce the production of acetic acid byproducts in prokaryotic cells through long-term research.
[0006] Unlike previously reported traditional fermentation process improvement or genetic modification strategies, the technical solution of this application overcomes the shortcomings of existing technologies. From the perspective of targeted genetic modification of strains, it provides an innovative strategy that is simple, effective, and widely applicable to improve the problem of acetic acid production, thereby guiding the optimization and modification of chassis host cells and the improvement of fermentation processes. It has important theoretical significance and industrial application value.
[0007] On one hand, this disclosure provides a recombinant prokaryotic cell modified by directed genetic engineering to include defects in one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, wherein the recombinant prokaryotic cell produces less acetic acid compared to the unmodified prokaryotic cell.
[0008] On the other hand, this disclosure provides a method for preparing recombinant prokaryotic cells as described herein, the method comprising directed genetic engineering of prokaryotic cells to produce defects in one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, wherein the recombinant prokaryotic cells produce less acetic acid compared to unmodified prokaryotic cells.
[0009] On the other hand, this disclosure provides a product comprising the recombinant prokaryotic cells described herein.
[0010] On the other hand, this disclosure provides the use of the recombinant prokaryotic cells, methods, or products described herein in metabolic engineering; said metabolic engineering includes recombinant protein expression, plasmid production, and / or peptide production, wherein acetic acid production is reduced during metabolic engineering.
[0011] On the other hand, this disclosure provides a recombinant production method, comprising: providing the recombinant prokaryotic cells described herein as recombinant production host cells, or performing targeted genetic engineering on the recombinant production host cells by the methods described herein, wherein the recombinant production host cells contain a target product coding sequence; and culturing the host cells under conditions suitable for producing the target product.
[0012] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this invention. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description
[0013] The present invention will be further described below with reference to the accompanying drawings, which are shown only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.
[0014] Figure 1 A schematic diagram of gene mutation using the λRed recombination system is shown.
[0015] Figure 2 The gene annotations of DNA regions that influence the production of the metabolic byproduct acetic acid are shown.
[0016] Figure 3 The DNA region where deletion mutations in E. coli B affect the production of the metabolic byproduct acetic acid was shown in the subsequent PCR verification of the strain.
[0017] Figure 4The DNA region in E. coli W3110 strain where deletion mutations affect the production of the metabolic byproduct acetic acid was shown by PCR verification of the strain.
[0018] Figure 5 The study shows a comparison of acetic acid production in E. coli B strain and its corresponding mutant strain in a medium with glucose as the main carbon source.
[0019] Figure 6 The study compared the acetic acid production of E. coli B strain and its corresponding mutant strain in a medium with glycerol as the main carbon source.
[0020] Figure 7 The study shows a comparison of acetic acid production in E. coli W3110 strain and its corresponding mutant strain in a medium with glucose as the main carbon source.
[0021] Figure 8 The study shows a comparison of acetic acid production in E. coli W3110 strain and its corresponding mutant strain in a medium with glycerol as the main carbon source. Detailed Implementation
[0022] All numerical ranges provided herein are intended to clearly include all values falling between the endpoints of the range and the range of values between them. In this application, any numerical value, whether or not it is modified by words such as "about," encompasses an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this application, each "implementation" equally refers to and covers implementations of the methods and systems of this application. In this application, one or more technical features in any implementation can be freely combined with one or more technical features in any one or more other implementations, and the resulting implementations are also part of the disclosure of this application. Features mentioned in this invention or features mentioned in the embodiments can be combined. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.
[0023] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”
[0024] The technical solution of this application overcomes the shortcomings of the prior art. By performing targeted genetic modification of prokaryotic cells, it provides an innovative strategy for solving the problem of acetic acid production that is simple, effective, and widely applicable. The technical solution of this invention can guide the optimization and modification of chassis host cells and the improvement of fermentation processes, and has significant theoretical and industrial application value.
[0025] On one hand, this disclosure provides a recombinant prokaryotic cell modified by directed genetic engineering to include defects in one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, wherein the recombinant prokaryotic cell produces less acetic acid compared to the unmodified prokaryotic cell.
[0026] In some embodiments, the gene defects in the recombinant prokaryotic cells described in this application include: the absence of DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and / or pgl in the genome. In some embodiments, the gene defects in the recombinant prokaryotic cells include: failure to transcribe RNA encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and / or pgl. In some embodiments, the gene defects in the recombinant prokaryotic cells include: failure to express proteins encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and / or pgl, or the expressed proteins encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and / or pgl are unable to perform their biological functions or are lost. In some embodiments, the gene defects in the recombinant prokaryotic cells include the inability to transport or secrete proteins encoded by the expressed galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, or pgl.
[0027] The term "gene defect" as used herein refers to a phenomenon in which a gene is unable to perform its normal function due to some reason. The reasons for this inability to perform normal gene function include, but are not limited to: gene knockout (e.g., knockout via the λ-Red system or CRISPR-Cas editing system based on homologous recombination), gene mutation (e.g., mutation via the λ-Red system or CRISPR-Cas editing system based on homologous recombination), gene knockdown (e.g., inhibiting gene transcription, inhibiting its mRNA translation, degrading its mRNA, or inducing gene silencing), or the absence or mutation of gene transcription regulators (e.g., promoters), or other causes that reduce the content of gene products and / or weaken and / or disrupt their function and / or activity.
[0028] In this article, "galK" refers to the galactokinase gene. "galT" refers to the galactose-1-phosphate uridine transferase gene. "galE" refers to the UDP-galactose-4-epimerase gene or UDP-glucose-4-epimerase gene, which encodes UDP-galactose-4-epimerase, an enzyme that catalyzes the reversible conversion of UDP-glucose to UDP-galactose in normal metabolic pathways. In prokaryotic cell genomes (e.g., *E. coli*), the three genes galK, galT, and galE (or "galKTE") belong to the same polycistronic promoter and are co-transcribed by a common upstream promoter. The "promoter of galK-galT-galE" refers to the common promoter of the polycistronic galK-galT-galE. "modA" refers to the molybdate-binding protein gene. "modB" refers to the membrane channel protein gene of the molybdate transport system. In this article, "modC" and "modF" refer to ATP-binding protein genes of the molybdate transport system. "modE" encodes a transcriptional regulator that can influence the synthesis of the three proteins mentioned above: "modA," "modB," and "modC." "acrZ" refers to a multidrug efflux pump accessory protein gene. "ybhA" refers to a pyridoxal 5'-phosphate (PLP) phosphatase gene. "pgl" refers to a 6-phosphogluconolactonease gene.
[0029] The recombinant prokaryotic cells according to this disclosure exhibit improved acetic acid production. In some embodiments, the improved acetic acid production refers to a reduction in the production of acetic acid, a metabolic byproduct, during fermentation. In some embodiments, the improved acetic acid production refers to an improved trend in the production of acetic acid, a metabolic byproduct, during fermentation.
[0030] The term "acetic acid" as used in the context of this article is intended to encompass all substances containing the acetate ion (C2H3O2). - Compounds containing an acetic acid group or an acetic acid group, including acetic acid, acetates or their ester forms and derivatives thereof.
[0031] In some embodiments, the targeted genetic engineering described herein includes knocking out one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the knockout includes full-length knockout or partial knockout. In some embodiments, the targeted genetic engineering described herein includes introducing mutations into sequences encoding one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes to render them unable to perform their biological functions or to result in loss of activity. In some embodiments, the mutations described herein include insertions, deletions, or substitutions. The term "mutation" includes, but is not limited to, frameshift mutations, whole-frame mutations, nonsense mutations, and missense mutations, which result in a gene failing to encode a complete protein, or resulting in a protein with a sequence different from the wild type, or resulting in a reduced or lost activity of the protein. In some embodiments, the directed genetic engineering described herein includes introducing mutations in one or more transcriptional regulatory regions of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the directed genetic engineering described herein includes introducing mutations in one or more promoter regions of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the directed genetic engineering described herein includes knocking out one or more transcriptional regulatory regions of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the directed genetic engineering described herein includes knocking out one or more promoters of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. The term "promoter" refers to a polynucleotide sequence that allows transcription of a target gene and regulates its expression. The promoter includes a sequence recognizable by RNA polymerase and a transcription start site. In some embodiments, the promoter is preferably a specific promoter, such as a promoter regulating galK-galT-galE transcription. In some embodiments, the promoter is preferably a specific promoter, such as a promoter regulating modF-modE and acrZ transcription.In some embodiments, the promoter is preferably a specific promoter, such as a promoter regulating modA-modB-modC transcription. In some embodiments, the promoter is preferably a specific promoter, such as a promoter regulating ybhA and pgl transcription. In some embodiments, the promoter can be any combination of the aforementioned specific promoters. The "promoter regulating galK-galT-galE transcription" described herein refers to a common promoter of polycistronic galK-galT-galE, located in the intergenic region of modF and galE in the genome. The "promoter regulating modF-modE and acrZ transcription" described herein is located in the intergenic region of modE and acrZ in the genome. The "promoter regulating modA-modB-modC transcription" described herein is located in the intergenic region of modA and acrZ in the genome. The "promoter regulating ybhA and pgl transcription" described herein is located in the intergenic region of ybhA and pgl in the genome. In some embodiments, the sequence of the promoter region regulating galK-galT-galE transcription described herein is shown in SEQ ID NO:17. In some embodiments, the promoters regulating modF-modE and acrZ transcription are shown in SEQ ID NO:20. In some embodiments, the promoters regulating modA-modB-modC transcription are shown in SEQ ID NO:21. In some embodiments, the promoters regulating ybhA and pgl transcription are shown in SEQ ID NO:22. In some embodiments, the directed genetic engineering described herein includes introducing mutations in the region regulating the transport or secretion of proteins encoded by one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes to interfere with their transport or secretion. The “region regulating transport or secretion” includes a signal peptide sequence or guide peptide sequence. Prokaryotic signal peptide pathways include the Sec pathway, Tat pathway, etc. A signal peptide sequence or guide peptide sequence exists at one end of a protein, which encodes a signal peptide or guide peptide that guides the protein to localize, transport, and secrete. If the signal peptide sequence or guide peptide sequence regulating the localization, transport, or secretion of a protein is mutated or knocked out, the protein's biological function will be affected. In some embodiments, the targeted genetic engineering described herein includes suppressing the expression of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes using RNA interference technology. In specific embodiments, the RNA interference technology includes, but is not limited to, interference using siRNA or interference using shRNA.
[0032] In the context of this application, the exemplary structure of the DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl in the prokaryotic genome is as follows: Figure 2 As shown. Figure 2 The arrows indicate the orientation of each gene. The inventors discovered for the first time that this DNA region is closely related to the production of the metabolic byproduct acetic acid. Further analysis of this acid-producing region revealed that the three genes galK-galT-galE (or "galKTE" genes) belong to the same polycistronic gene and are co-transcribed by a common upstream promoter (the intergenic region between modF and galE). Knocking out this promoter blocks the transcription and expression of these three genes. As an example, embodiments of this disclosure demonstrate that a knockout strategy involving the near-full-length region enclosed by the galK-galT-galE promoter and a portion of the pgl coding region can improve acetic acid production. Based on the results of the embodiments, the structure of the acid-producing region, and common knowledge in the art, those skilled in the art can reasonably expect that partially or completely knocking out the coding sequences of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl proteins and / or knocking out or mutating one or more of their corresponding promoters and other regulatory elements can achieve the same effect of removing protein function.
[0033] In some embodiments, the directed genetic engineering described herein includes (i) knocking out or mutating the promoter of galK-galT-galE and / or knocking out or mutating the galK, galT, and galE genes; or (ii) knocking out or mutating the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii) knocking out or mutating the promoter of pgl and / or knocking out or mutating the pgl gene; or any combination of (i), (ii), and (iii), wherein the knockout includes partial or full-length knockout, and the mutation includes insertion, deletion, or substitution. In some embodiments, the directed genetic engineering described herein includes knocking out or mutating the promoter of galK-galT-galE, and knocking out or mutating the modF, modE, acrZ, modA, modB, modC, and ybhA genes, and completely or partially knocking out or mutating the pgl gene. In some embodiments, the gene defects described herein include (i') partial or complete deletion of the galK-galT-galE promoter and / or the galK, galT, and galE genes; or (ii') partial or complete deletion of the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii') partial or complete deletion of the pgl promoter and / or the pgl gene; or any combination of (i), (ii), and (iii). In some embodiments, the gene defects described herein include non-transcription of galK-galT-galE, the absence of DNA sequences encoding modF, modE, acrZ, modA, modB, modC, and ybhA in the genome, and partial or complete deletion of the DNA sequence encoding pgl or its promoter sequence in the genome. In some embodiments, the DNA sequence encoding the full-length pgl gene is shown in SEQ ID NO:23. In some exemplary embodiments, the partially deleted DNA sequence encoding pgl may include the sequence shown in SEQ ID NO:18. In some embodiments, the genetic defects described herein include the absence of a sequence in the genome as shown in SEQ ID NO:19 or a sequence having at least 70% sequence identity with SEQ ID NO:19.
[0034] In some embodiments, the amino acid sequence of the protein encoded by galK described herein is shown in SEQ ID NO:6. In some embodiments, the amino acid sequence of the protein encoded by galT described herein is shown in SEQ ID NO:7. In some embodiments, the amino acid sequence of the protein encoded by galE described herein is shown in SEQ ID NO:8. In some embodiments, the amino acid sequence of the protein encoded by modF described herein is shown in SEQ ID NO:9. In some embodiments, the amino acid sequence of the protein encoded by modE described herein is shown in SEQ ID NO:10. In some embodiments, the amino acid sequence of the protein encoded by acrZ described herein is shown in SEQ ID NO:11. In some embodiments, the amino acid sequence of the protein encoded by modA described herein is shown in SEQ ID NO:12. In some embodiments, the amino acid sequence of the protein encoded by modB described herein is shown in SEQ ID NO:13. In some embodiments, the amino acid sequence of the protein encoded by modC described herein is shown in SEQ ID NO:14. In some embodiments, the amino acid sequence of the protein encoded by ybhA described herein is shown in SEQ ID NO:15. In some embodiments, the amino acid sequence of the protein encoded by pgl described herein is shown in SEQ ID NO:16. In some embodiments, the amino acid sequence of the protein encoded by each gene galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, or pgl described herein includes a homologous sequence having at least 70% sequence identity with the above sequences, wherein the protein having the homologous sequence can achieve equivalent biological function or activity.
[0035] On the other hand, this disclosure provides a method for preparing the recombinant prokaryotic cells described herein, the method comprising directing genetic engineering the prokaryotic cells to produce defects in one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl; wherein the recombinant prokaryotic cells produce less acetic acid compared to the unmodified prokaryotic cells.
[0036] In some embodiments, the targeted genetic engineering described herein includes knocking out one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl. In some embodiments, the knockout includes full-length knockout or partial knockout. In some embodiments, the targeted genetic engineering described herein includes introducing mutations into the DNA sequence encoding one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes to render them unable to perform their biological function or to cause loss of activity. In some embodiments, the mutations described herein include insertions, deletions, or substitutions. In some embodiments, the targeted genetic engineering described herein includes introducing mutations into one or more transcriptional regulatory regions of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the genetic engineering described herein includes introducing mutations in one or more promoter regions of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the targeted genetic engineering described herein includes knocking out one or more transcriptional regulatory regions of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the targeted genetic engineering described herein includes knocking out one or more promoters of one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes. In some embodiments, the promoter is preferably a specific promoter, such as a promoter regulating galK-galT-galE transcription, a promoter regulating modF-modE and acrZ transcription, a promoter regulating modA-modB-modC transcription, and / or a promoter regulating ybhA and pgl transcription. In some embodiments, the promoter can be any combination of the above-mentioned specific promoters. In some embodiments, the sequence of the promoter region described herein includes SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:21, and / or SEQ ID NO:22.In some embodiments, the sequence of the promoter region described herein includes a sequence having at least 90%, at least 85%, at least 80%, at least 75%, and at least 70% identity with SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:21, and / or SEQ ID NO:22, respectively. In some embodiments, the directed genetic engineering described herein includes introducing mutations in a region regulating the transport or secretion of proteins encoded by one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes to interfere with their transport or secretion. In the context of this application, the "region regulating the transport or secretion of proteins" includes a signal peptide sequence or a guide peptide sequence. In some embodiments, the directed genetic engineering described herein includes inhibiting the expression of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes using RNA interference technology. In specific implementations, the RNA interference technology includes, but is not limited to, interference using siRNA or interference using shRNA.
[0037] In some embodiments, the amino acid sequence of the protein encoded by galK described herein is shown in SEQ ID NO:6. In some embodiments, the amino acid sequence of the protein encoded by galT described herein is shown in SEQ ID NO:7. In some embodiments, the amino acid sequence of the protein encoded by galE described herein is shown in SEQ ID NO:8. In some embodiments, the amino acid sequence of the protein encoded by modF described herein is shown in SEQ ID NO:9. In some embodiments, the amino acid sequence of the protein encoded by modE described herein is shown in SEQ ID NO:10. In some embodiments, the amino acid sequence of the protein encoded by acrZ described herein is shown in SEQ ID NO:11. In some embodiments, the amino acid sequence of the protein encoded by modA described herein is shown in SEQ ID NO:12. In some embodiments, the amino acid sequence of the protein encoded by modB described herein is shown in SEQ ID NO:13. In some embodiments, the amino acid sequence of the protein encoded by modC described herein is shown in SEQ ID NO:14. In some embodiments, the amino acid sequence of the protein encoded by ybhA described herein is shown in SEQ ID NO:15. In some embodiments, the amino acid sequence of the protein encoded by pgl described herein is shown in SEQ ID NO:16. In some embodiments, the amino acid sequence of the protein encoded by each gene galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, or pgl described herein includes a homologous sequence having at least 70% sequence identity with the above sequences, wherein the protein having the homologous sequence can achieve equivalent biological function or activity.
[0038] Due to interspecies differences, the amino acid sequences of proteins encoded by galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, or pgl in different prokaryotic cells may differ from the sequences shown in SEQ ID NO:6-16, respectively. The scope of protection of this application is intended to cover homologous sequences having at least 90%, 85%, 80%, 75%, and 70% identity with the sequences shown in SEQ ID NO:6-16, respectively, and whose encoded proteins can perform the same or equivalent biological functions or activities as the proteins encoded by galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, or pgl listed herein. Therefore, the technical solution of directed genetic engineering modification of the homologous sequences can achieve the technical effect of reducing acetic acid production described in this application, and is thus also covered within the scope of protection claimed in this application.
[0039] In some embodiments, the DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl described herein include SEQ ID NO:1. In some embodiments, the DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl described herein include sequences having at least 70% sequence identity with SEQ ID NO:1. Due to interspecies differences and codon degeneracy, the DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl described herein may differ from SEQ ID NO:1. The scope of protection of this application is intended to cover homologous sequences having at least 90%, 85%, 80%, 75%, and 70% identity with the sequence shown in SEQ ID NO:1, and whose encoded proteins can perform the same or equivalent biological functions or activities as those encoded by galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl listed herein. Therefore, the technical solution of targeted genetic engineering modification of the homologous sequences can achieve the technical effect of reducing acetic acid production described in this application, and is thus also covered within the scope of protection claimed in this application.
[0040] In some embodiments, the directed genetic engineering described herein includes (i) knocking out or mutating the galK-galT-galE promoter and / or knocking out or mutating the galK, galT, and galE genes; or (ii) knocking out or mutating the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii) knocking out or mutating the pgl promoter and / or knocking out or mutating the pgl gene; or any combination of (i), (ii), and (iii), wherein the knockout includes partial or full-length knockout, and the mutation includes insertion, deletion, or substitution. In some embodiments, the gene defect described herein includes knocking out or mutating the galK-galT-galE promoter, knocking out or mutating the modF, modE, acrZ, modA, modB, modC, and ybhA genes, and completely or partially knocking out or mutating the pgl gene or the promoter regulating pgl transcription. In some embodiments, the promoter regulating pgl transcription refers to the promoter regulating ybhA and pgl transcription. For example, in some embodiments, the promoter sequence regulating galK-galT-galE transcription is shown in SEQ ID NO:17. For example, in some embodiments, the promoter sequence regulating ybhA and pgl transcription is shown in SEQ ID NO:22. In some embodiments, the "knockout" described in this invention can be a full-length knockout or a partial knockout. For example, in a specific embodiment, knocking out the pgl gene can be a full-length knockout or a partial knockout. For example, in a more specific embodiment, the sequence of the partially knocked-out pgl gene can be as shown in SEQ ID NO:18. For example, in some embodiments, the directed genetic engineering modification described herein includes knockout or deletion mutations such as the sequence shown in SEQ ID NO:19 or sequences having at least 90%, 85%, 80%, 75%, or 70% identity with SEQ ID NO:19.
[0041] In some embodiments, the targeted genetic engineering described in this application is performed using gene editing technology. For example, the λ-Red system or the CRISPR-Cas editing system is used. For example, in a specific embodiment, the λRed recombination system is used to perform partial or full-length deletion mutations in the galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl regions of the genome. For example, in a specific embodiment, the λRed recombination system is used to perform partial or full-length deletion mutations in the sequence shown in SEQ ID NO:19 or a sequence having at least 90%, 85%, 80%, 75%, or 70% identity with SEQ ID NO:19.
[0042] In some embodiments, the prokaryotic cells described herein include *Escherichia coli* cells. In some embodiments, the *Escherichia coli* described herein includes strains B and strain K. In some embodiments, the strains K include strains K12. In some embodiments, the *Escherichia coli* described herein includes TOP10, TOP10B, W3110, W3110(DE3), DH5α, MG1655, and / or DH10b and their derived strains. In some embodiments, the recombinant *Escherichia coli* cells described herein are grown, proliferated, or fermented in a medium with glycerol as the primary carbon source. In some embodiments, the recombinant *Escherichia coli* cells described herein are grown, proliferated, or fermented in a medium with glucose as the primary carbon source.
[0043] The terms "chassis host," "chassis host cell," or "chassis cell" as used herein refer to the host cell in which the metabolic reaction occurs. This application utilizes prokaryotic cells as chassis cells and modifies them using gene editing technology to obtain excellent cell factories for the efficient synthesis of target products, providing a new approach for the construction of industrial microbial chassis in the future. Those skilled in the art should understand that the chassis cells applicable to the technical solution of this application include wild-type prokaryotic cell lines as well as mature prokaryotic cell lines that have already been used as cell factories but still require modification to address the generation of acetic acid byproducts. The targeted genetic modification of mature prokaryotic cells described in this application can easily and efficiently reduce acetic acid generation without the need for de novo screening and modification. Therefore, compared with existing technologies, the technical solution of this application provides an innovative strategy with wide applicability, ease of implementation, and significant effects in solving the problem of acetic acid production.
[0044] On the other hand, this disclosure provides a product comprising the recombinant prokaryotic cells described herein.
[0045] On the other hand, this disclosure provides the use of the recombinant prokaryotic cells, methods, or products described herein in metabolic engineering, which includes recombinant protein expression, plasmid production, and / or peptide production, wherein acetic acid production is reduced during metabolic engineering. In some embodiments, the recombinant protein expression includes the production of recombinant protein drugs, including but not limited to peptide hormones, cytokines, and recombinant enzymes.
[0046] On the other hand, this disclosure provides a recombinant production method, comprising: providing the recombinant prokaryotic cells described herein as recombinant production host cells, or performing directed genetic engineering on the recombinant production host cells as described herein, wherein the recombinant production host cells contain a target product coding sequence; and culturing the host cells under conditions suitable for producing the target product. In some embodiments, the culture includes a plate culture step, a shaker culture step, a scale-up culture step, a cell bank preparation step, and / or a fermentation culture step. In some embodiments, the culture includes small-scale culture, pilot-scale culture, large-scale culture, process validation culture, and / or industrial production culture. In some embodiments, the culture further includes providing a carbon source, such as sugars, organic acids, alcohols, lipids, hydrocarbons, carbonates, carbon dioxide (for autotrophic microorganisms), amino acids, etc., as the main carbon source. A substance that provides carbon to microorganisms during their growth is called a carbon source. In the process of recombinant production using microorganisms, a culture medium containing a carbon source is required for the microorganisms; the carbon source with the highest content in the culture medium is the main carbon source. In some embodiments, the culture uses sugars or alcohols as the main carbon source, for example, glucose or glycerol. For example, in some embodiments, the culture uses carbohydrates as the primary carbon source. The carbohydrates include glucose, fructose, maltose, sucrose, starch, galactose, lactose, mannose, cellobiose, cellulose, hemicellulose, chitin, or lignin, etc. For example, in some embodiments, the culture uses glucose as the primary carbon source. For example, in some embodiments, the culture uses alcohols as the primary carbon source. The alcohols include ethanol, glycerol, etc. For example, in some embodiments, the culture uses glycerol as the primary carbon source. In some embodiments, the culture further includes providing a supplementary culture medium with carbohydrates or alcohols as the primary carbon source. In some embodiments, the technical solution of this application further includes adding or supplementing the supplementary culture medium with glucose or glycerol as the primary carbon source.
[0047] In some embodiments, the target product includes recombinant proteins, plasmids, and / or peptides. In some embodiments, the target product includes recombinant protein drugs, including but not limited to peptide hormones, cytokines, and recombinases.
[0048] In some embodiments, the acetic acid production process is improved, which includes an improved trend in the production of the metabolic byproduct acetic acid or a reduction in the amount of acetic acid produced, for example, a reduction of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% in acetic acid production.
[0049] In some embodiments, the reduction in the amount of acetic acid produced during the recombinant production process is compared to that of the corresponding pre-modified strain (or wild-type strain). In some embodiments, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 20-50%. In a specific embodiment, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 30%. In some embodiments, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 50-80%. In a specific embodiment, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 62%. In some embodiments, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 80-99.9%. In a specific embodiment, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 97%. In a specific embodiment, the amount of acetic acid produced by the directionally genetically modified recombinant prokaryotic cells is reduced by 99.7%.
[0050] In some embodiments, the culture includes culturing recombinant prokaryotic cells containing the directed genetically engineered strain described in this application in a medium with glucose as the primary carbon source. In some embodiments, the culture includes culturing recombinant prokaryotic cells containing the directed genetically engineered strain described in this application in a medium with glycerol as the primary carbon source. In some embodiments, the culture includes culturing E. coli B mutant strains containing the directed genetically engineered strain described in this application in a medium with glucose as the primary carbon source. In some embodiments, the culture includes culturing E. coli B mutant strains containing the directed genetically engineered strain described in this application in a medium with glycerol as the primary carbon source. In some embodiments, the culture includes culturing E. coli W3110 mutant strains containing the directed genetically engineered strain described in this application in a medium with glucose as the primary carbon source. In some embodiments, the culture includes culturing E. coli W3110 mutant strains containing the directed genetically engineered strain described in this application in a medium with glycerol as the primary carbon source.
[0051] This application innovatively discovers a DNA region in the genome that significantly affects the production of acetic acid, a metabolic byproduct of *E. coli*. This DNA region primarily encodes galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, and verifies the surprising technical effects of this region. Using *E. coli* as an example, this paper first uses gene editing technology to perform deletion mutations on the aforementioned DNA region; then, it evaluates and compares the acetic acid production trends and yields of the corresponding mutant strains and the unmodified wild-type strains, finding that the acetic acid production trend of the mutant strains is significantly improved compared to the unmodified wild-type strains, and the acetic acid yield is significantly reduced. Based on this disclosure, those skilled in the art will understand that, in addition to wild-type strains serving as starting strains, existing host cells used for recombinant production can also be optimized using the modification strategy of this application to address the acid production problem. For example, the host W3110(DE3), which is already used in the industry to produce recombinant proteins, can be further modified using the targeted genetic engineering of this application to improve acid production, thereby making it a better expression host. Such modification and application are also included within the scope of protection of this application. In summary, the modification strategy provided in this application has potential for wide-ranging and universal applications in metabolic engineering, making an outstanding contribution to the prior art.
[0052] The inventors have discovered for the first time that this DNA region is closely related to the production of the metabolic byproduct acetic acid. The strategy based on this region, as presented herein, can significantly improve acetic acid production. It is simple, effective, and widely applicable, and is suitable for both E. coli B and K12 series strains. Taking E. coli B strain and K12 series strain W3110 as examples, this application, through gene editing technology, mutated the above-mentioned DNA region to a full length and found that in culture media using glucose or glycerol as the main carbon source, the acetic acid production of the corresponding mutant strains was significantly reduced compared to the original strain, and the acetic acid production trend was also greatly improved. Specifically, in culture media using glycerol as the main carbon source, acetic acid production of the E. coli B mutant and W3110 mutant was reduced by approximately 97% and approximately 99.7%, respectively; in culture media using glucose as the main carbon source, acetic acid production of the E. coli B mutant and W3110 mutant was reduced by approximately 30% and approximately 62%, respectively.
[0053] Example
[0054] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.
[0055] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.
[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to one skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this application. The preferred embodiments and materials described herein are for illustrative purposes only.
[0057] Example 1. Deletion mutations in DNA regions of E. coli B and W3110 that significantly affect the production of the metabolic byproduct acetic acid. Change
[0058] (1) Using the λRed recombination system ( Figure 1 ) Target region deletion mutation: Based on the genomic sequence of the corresponding strain, primers SEQ ID NO:2 / SEQ ID NO:3 (synthesized by Suzhou Genewise Biotechnology Co., Ltd.) containing 30bp homologous arms were designed for the DNA region (SEQ ID NO:1) that affects the production of the metabolic byproduct acetic acid. Using plasmid pKD13 (purchased from HonorGene, catalog number HG-VJC0520; RED homologous recombination resistance fragment template plasmid, which can be replaced by any other equivalent plasmid) as a template, the mutant resistance fragment FRT-kanamycin resistance gene element-FRT was amplified.
[0059] (2) The above-mentioned resistance fragment elements were electroporated into E. coli B or W3110 electroporated competent cells containing plasmid pKD46 (purchased from HonorGene, catalog number HG-VJC0521, a gene knockout vector for Red homologous recombination, which can be replaced by any other equivalent plasmid), plated on LB solid plates containing kanamycin, and cultured upside down overnight to obtain single clones.
[0060] (3) The above clones were verified by PCR using the outer primers of the homologous arm SEQ ID NO:4 / SEQ ID NO:5 to obtain positive clones (the mutant strain with the deletion of the region to be modified after fragment recombination).
[0061] Example 2. Preparation of culture medium for evaluating the formation of the metabolic byproduct acetic acid
[0062] (1) Solution 1 preparation: Prepare according to 25.6 g / L disodium hydrogen phosphate heptahydrate, 6 g / L potassium dihydrogen phosphate, 2 g / L ammonium chloride, 1 g / L sodium chloride, and 4 g / L yeast powder. After preparation, sterilize at 121℃ for 20 minutes.
[0063] (2) Solution 2 preparation: Prepare with 20g / L glucose and 0.5g / L magnesium sulfate. After preparation, sterilize at 121℃ for 20 minutes.
[0064] (3) Solution 3 preparation: Prepare with 20g / L glycerol and 0.5g / L magnesium sulfate. After preparation, sterilize at 121℃ for 20 minutes.
[0065] (4) When using, mix solutions 1 and 2 at a ratio of 1:1 (V / V) to obtain culture medium A with glucose as the main carbon source; mix solutions 1 and 3 at a ratio of 1:1 (V / V) to obtain culture medium B with glycerol as the main carbon source.
[0066] Example 3. Assessment of acetic acid production in E. coli B and W3110 related strains
[0067] (1) Inoculate the wild-type strain of E. coli B, the wild-type strain of W3110 and the corresponding mutant strain obtained in Example 1 into LB liquid medium and culture overnight at 30°C and 220 rpm for 16-18 h.
[0068] (2) Using the overnight culture from (1) as seed cells, according to the initial OD 600 The 0.05 was added to 250 mL shake flasks containing 25 mL of culture medium (one flask each of A and B culture media from Example 2), and then incubated at 30 °C and 220 rpm.
[0069] (3) At incubation time points of 6, 9, 10, and 24 hours, 1 mL of culture was taken each time, and the OD was measured. 600 The sample was centrifuged (12000 rpm, 15 min) and the supernatant was collected. The acetate concentration of the supernatant sample was then determined using a kit (Roche, catalog number 07395485001) to calculate the acetic acid production. Results are as follows: Figure 5-8 As shown.
[0070] Figure 5 The results showed that, in a culture medium with glucose as the main carbon source, the E. coli B mutant strain produced less acetic acid compared to the wild-type E. coli B strain. Furthermore, the mutation did not affect the strain's growth; in fact, it promoted the strain's growth compared to the wild-type B strain.
[0071] Figure 6 The results showed that, in a culture medium with glycerol as the main carbon source, the E. coli B mutant strain produced significantly less acetic acid compared to the wild-type E. coli B strain. Meanwhile, the mutation did not affect the strain's growth.
[0072] Figure 7The results showed that in a culture medium with glucose as the main carbon source, the E. coli W3110 mutant strain produced significantly less acetic acid compared to the wild-type E. coli W3110 strain. Furthermore, the mutation did not affect the strain's growth; in fact, it promoted the strain's growth compared to the wild-type W3110 strain.
[0073] Figure 8 The results showed that in a medium with glycerol as the main carbon source, the E. coli W3110 mutant strain produced significantly less acetic acid compared to the wild-type strain. Meanwhile, the mutation did not affect the strain's growth; in fact, it promoted the strain's growth compared to the wild-type W3110 strain.
[0074] In summary, the percentage decrease in acetic acid production of each mutant strain at the 24-hour time point compared to the corresponding wild-type strain is summarized in Table 1.
[0075] Table 1. Summary of the percentage decrease in acetic acid production for each mutant strain at 24 h.
[0076]
[0077] Example 4. Recombinant Production / Recombinant Protein Expression Experiment
[0078] (1) The coding sequence of the recombinant protein was ligated into the pET28a expression vector by gene synthesis (Suzhou Genewise Biotechnology Co., Ltd.) to obtain the corresponding expression plasmid.
[0079] (2) The above expression plasmid was transformed into the W3110-derived mutant strain, cultured on LB solid plates containing kanamycin, and positive clones were screened.
[0080] (3) Positive clones were inoculated into 15 mL of medium B containing glycerol with kanamycin as the main carbon source and cultured at 30℃ and 220 rpm until the OD600 reached 0.6-1.0. Then, IPTG with a final concentration of 0.2 mM was added and expression was induced at 20℃ for 16-20 h.
[0081] Appendix: The serial numbers and specific sequences described in this application are shown below.
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
Claims
1. A recombinant prokaryotic cell modified by directed genetic engineering to include defects in one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, wherein the recombinant prokaryotic cell produces less acetic acid compared to the unmodified prokaryotic cell.
2. The recombinant prokaryotic cell of claim 1, wherein the gene defect includes: (1) The genome does not contain DNA sequences encoding galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and / or pgl; (2) It does not transcribe RNAs of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and / or pgl; (3) The expression does not express proteins encoded by galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and / or pgl, or the expressed proteins encoded by galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and / or pgl are unable to perform their biological functions or are inactive; and / or (4) The proteins encoded by the expressed galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and / or pgl cannot be transported or secreted.
3. The recombinant prokaryotic cell as described in claim 1, wherein the directed genetic engineering modification includes: (a) Knockout of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes, wherein the knockout includes full-length knockout or partial knockout; (b) Introducing a mutation into a DNA sequence encoding one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; (c) Introducing a mutation in one or more transcriptional regulatory regions (including one or more promoter regions) of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; (d) Knock out one or more transcriptional regulatory regions (including one or more promoter regions) of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and pgl genes, wherein the knockout includes full-length knockout or partial knockout; (e) Introducing a mutation in a region regulating the transport or secretion of a protein encoded by one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; and / or (f) Inhibit the expression of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes by RNA interference.
4. The recombinant prokaryotic cell as described in claim 1, wherein the directed genetic engineering modification includes: (i) Knockout or mutation of the promoter of galK-galT-galE and / or knockout or mutation of the galK, galT and galE genes; or (ii) Knockout or mutation of the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii) Knockout or mutation of the pgl promoter and / or knockout or mutation of the pgl gene; or Any combination of (i), (ii), and (iii) The knockout includes partial or full-length knockout, and the mutation includes insertion, deletion, or substitution.
5. The recombinant prokaryotic cell of claim 1, wherein the gene defect includes: (i') partial or complete deletion of the galK-galT-galE promoter and / or the galK, galT, and galE genes; or (ii') partial or complete deletion of the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii') partial or complete deletion of the pgl promoter and / or pgl gene; or Any combination of (i'), (ii'), and (iii').
6. A method for preparing recombinant prokaryotic cells as described in any one of claims 1 to 5, the method comprising performing directed genetic engineering on the prokaryotic cells to produce defects in one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl; wherein the recombinant prokaryotic cells produce less acetic acid compared to the unmodified prokaryotic cells.
7. The method of claim 6, wherein the directed genetic engineering includes: (a) Knockout of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl, wherein the knockout includes full-length knockout or partial knockout; (b) Introducing a mutation into a DNA sequence encoding one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; (c) Introducing a mutation in one or more transcriptional regulatory regions (including one or more promoter regions) of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; (d) Knock out one or more transcriptional regulatory regions (including one or more promoter regions) of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and pgl genes, wherein the knockout includes full-length knockout or partial knockout; (e) Introducing a mutation in a region regulating the transport or secretion of a protein encoded by one or more genes selected from the group consisting of: galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes; said mutation includes insertion, deletion, or substitution; and / or (f) Inhibit the expression of one or more genes selected from the group consisting of galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA, and pgl genes by RNA interference.
8. The recombinant prokaryotic cells according to any one of claims 1 to 5, or the method according to claim 6 or 7, wherein... The amino acid sequence of the protein encoded by galK is shown in SEQ ID NO:
6. The amino acid sequence of the protein encoded by galT is shown in SEQ ID NO:
7. The amino acid sequence of the protein encoded by galE is shown in SEQ ID NO:
8. The amino acid sequence of the protein encoded by modF is shown in SEQ ID NO:
9. The amino acid sequence of the protein encoded by modE is shown in SEQ ID NO:
10. The amino acid sequence of the protein encoded by the acrZ is shown in SEQ ID NO:
11. The amino acid sequence of the protein encoded by modA is shown in SEQ ID NO:
12. The amino acid sequence of the protein encoded by modB is shown in SEQ ID NO:
13. The amino acid sequence of the protein encoded by modC is shown in SEQ ID NO:
14. The amino acid sequence of the protein encoded by ybhA is shown in SEQ ID NO:15, and / or The amino acid sequence of the protein encoded by the pgl is shown in SEQ ID NO:
16. Or homologous sequences that have at least 70% sequence identity with the above sequences, wherein, Proteins with the aforementioned homologous sequences can achieve equivalent biological functions or activities.
9. The recombinant prokaryotic cell of claim 3 or the method of claim 7, wherein the promoter comprises a promoter that regulates galK-galT-galE transcription, a promoter that regulates modF-modE and acrZ transcription, a promoter that regulates modA-modB-modC transcription and / or a promoter that regulates ybhA and pgl transcription.
10. The recombinant prokaryotic cell of claim 3 or the method of claim 7, wherein the promoter regulating galK-galT-galE transcription is shown in SEQ ID NO:17, the promoter regulating modF-modE and acrZ transcription is shown in SEQ ID NO:20, the promoter regulating modA-modB-modC transcription is shown in SEQ ID NO:21, and the promoter regulating ybhA and pgl transcription is shown in SEQ ID NO:
22.
11. The method of claim 7, wherein the directed genetic engineering comprises: (i) Knockout or mutation of the promoter of galK-galT-galE and / or knockout or mutation of the galK, galT and galE genes; or (ii) Knockout or mutation of the modF, modE, acrZ, modA, modB, modC, and ybhA genes; or (iii) Knockout or mutation of the pgl promoter and / or knockout or mutation of the pgl gene; or Any combination of (i), (ii), and (iii) The knockout includes partial or full-length knockout, and the mutation includes insertion, deletion, or substitution.
12. The method of claim 6 or 7, wherein the directed genetic engineering is performed using gene editing technology, such as the λ-Red system or the CRISPR-Cas editing system.
13. The recombinant prokaryotic cell of any one of claims 1 to 3 or the method of claim 6 or 7, wherein the DNA sequence encoding the galK, galT, galE, modF, modE, acrZ, modA, modB, modC, ybhA and pgl comprises SEQ ID NO:1 or a sequence having at least 70% sequence identity with SEQ ID NO:
1.
14. The recombinant prokaryotic cells according to any one of claims 1 to 5 or the method according to any one of claims 6 to 13, wherein the prokaryotic cells include Escherichia coli cells; The *Escherichia coli* strains include B-series and K-series strains, wherein the K-series strains include the K12 series strains. The *Escherichia coli* strains include TOP10, TOP10B, W3110, W3110(DE3), DH5α, MG1655 and / or DH10b and their derived strains.
15. A product comprising recombinant prokaryotic cells as described in any one of claims 1 to 5.
16. The use of the recombinant prokaryotic cells as described in any one of claims 1 to 5, or the method as described in any one of claims 6 to 14, or the product as described in claim 15, in metabolic engineering, wherein the metabolic engineering includes recombinant protein expression, plasmid production, and / or peptide production, wherein, Acetic acid production is reduced in metabolic engineering.
17. A method for recombinant production, comprising: Provides recombinant prokaryotic cells as described in any one of claims 1 to 5 as recombinant production host cells, or performs targeted genetic engineering on recombinant production host cells using the method described in any one of claims 6 to 13, wherein the recombinant production host cells contain a target product coding sequence; and The host cells are cultured under conditions suitable for producing the intended product.
18. The method of claim 17, wherein: The culture uses sugars or alcohols as the main carbon source, such as glucose or glycerol. The target product includes recombinant proteins, plasmids, and / or peptides; The improvement in acetic acid production during the recombinant production process includes an improved trend in the production of the metabolic byproduct acetic acid or a reduction in the amount of acetic acid produced, for example, a reduction of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9% in acetic acid production.