Search

CN-121991873-A - Recombinant engineering bacterium and application thereof in preparation of mannitol

CN121991873ACN 121991873 ACN121991873 ACN 121991873ACN-121991873-A

Abstract

The invention belongs to the technical field of bioengineering, and relates to a recombinant engineering bacterium and application thereof in preparing mannitol, wherein the recombinant engineering bacterium is a plasmid-free strain for producing cofactor NAD + by using cheap xylose and nicotinamide. The strain knocks out genes including ptsG encoding protein EIICB Glc in glucose PTS system, mtlA encoding EIICA protein in D-mannitol transmembrane transport, D-mannitol 1-phosphate dehydrogenase gene mtlD of D-mannitol utilization pathway, purine from the head synthesis regulatory protein purR and srlAE in sorbitol utilization gene cluster. The strain integrates genes of glucose transport facilitator glf, glucokinase glcK, xylose utilization gene cluster xylose isomerase xlyA, xylulophosphorylase xlyB, phosphoribosyl pyrophosphate synthetase prs, polyphosphate kinase ppk2, nicotinamide phosphoribosyl transferase nadV and nicotinamide mononucleotide adenylate transferase nadM in genome. The strain utilizes glucose and xylose for fermentation, and provides chassis bacteria for reactions requiring cofactor circulation.

Inventors

  • LI MIAN
  • YAN DONG
  • WANG YANNI
  • SHI MENG
  • WU YUSHUANG
  • DENG YANFENG

Assignees

  • 浙江华康药业股份有限公司

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A recombinant engineering bacterium is characterized in that the recombinant engineering bacterium takes escherichia coli as a host, the recombinant engineering bacterium knocks out genes including a gene ptsG encoding protein EIICB Glc in a glucose PTS system, a gene mtlA encoding EIICA protein in D-mannitol transmembrane transport and a gene mtlD of D-mannitol 1-phosphate dehydrogenase of a D-mannitol utilization way, and the genes including glucose transport assisting protein glf, glucose kinase glcK, xylose utilization gene cluster xylose isomerase xlyA and xylulophosphorylase xlyB are integrated in the genome of the recombinant engineering bacterium, The amino acid sequence of the glucose transport assisting protein glf is shown as SEQ ID NO.1, and the amino acid sequence of the glucokinase glcK is shown as SEQ ID NO. 2.
  2. 2. The recombinant engineering bacterium according to claim 1, wherein the nucleotide sequence of the glucose transport facilitator glf is shown in SEQ ID NO.3, and the nucleotide sequence of the glucokinase glcK is shown in SEQ ID NO. 4.
  3. 3. A recombinant engineering bacterium is characterized in that the recombinant engineering bacterium is preserved in China center for type culture collection (CCTCC NO: M20252967) in Wuhan in China on 12 months 19 days of 2025.
  4. 4. The recombinant engineering bacterium according to claim 3, wherein the recombinant engineering bacterium according to claim 1 is used as a chassis strain, wherein the recombinant engineering bacterium further knocks out srlAE of purine de novo synthesis regulatory protein purR and sorbitol utilization gene cluster, and the recombinant engineering bacterium further integrates genes including phosphoribosyl pyrophosphate synthetase prs, polyphosphate kinase ppk2, nicotinamide phosphoribosyl transferase nadV and nicotinamide mononucleotide adenylate transferase nadM into the genome, The amino acid sequence of the phosphoribosyl pyrophosphate synthetase prs is shown as SEQ ID NO.5, the amino acid sequence of the polyphosphate kinase ppk2 is shown as SEQ ID NO.7, the amino acid sequence of the nicotinamide phosphoribosyl transferase nadV is shown as SEQ ID NO.9, and the amino acid sequence of the nicotinamide mononucleotide adenylate transferase nadM is shown as SEQ ID NO. 11.
  5. 5. The recombinant engineering bacterium according to claim 4, wherein the nucleotide sequence of phosphoribosyl pyrophosphate synthetase is shown as SEQ ID NO.6, the nucleotide sequence of polyphosphate kinase ppk2 is shown as SEQ ID NO.8, the nucleotide sequence of nicotinamide phosphoribosyl transferase nadV is shown as SEQ ID NO.10, and the nucleotide sequence of nicotinamide mononucleotide adenylate transferase nadM is shown as SEQ ID NO. 12.
  6. 6. A recombinant engineering bacterium, characterized in that the recombinant engineering bacterium is used as a chassis strain, and is obtained by modifying recombinant engineering bacterium which is obtained by over-expressing mannitol dehydrogenase mdh and formate dehydrogenase fdh, wherein, The amino acid sequence of mannitol dehydrogenase mdh is shown as SEQ ID NO.13, and the amino acid sequence of formate dehydrogenase fdh is shown as SEQ ID NO. 14; the nucleotide sequence of mannitol dehydrogenase mdh is shown as SEQ ID NO.15, and the nucleotide sequence of formate dehydrogenase fdh is shown as SEQ ID NO. 16.
  7. 7. A fermentation method of the recombinant engineering bacterium according to claim 1, wherein the fermentation method comprises the steps of inoculating seed liquid after the activation of the recombinant engineering bacterium into a fermentation medium according to the inoculation amount of 1-5% by volume, and culturing at 37 ℃ with a carbon source of 5g/L glucose or 5g/L xylose or 2.5g/L glucose and xylose.
  8. 8. The method for fermenting recombinant engineering bacteria according to claim 7, wherein the fermentation medium comprises 5-20 g/L of corn steep liquor dry powder, 0.2~1g/L、K 2 HPO 4 12.54g/L、KH 2 PO 4 2.31g/L、MgSO 4 ·7H 2 O 1g/L -3 mL/L of ammonium citrate and trace element liquid, and the trace element liquid comprises the following components ：CaCl 2 0.5g/L、ZnSO 4 ·7H 2 O 0.18g/L、MnSO 4 ·H 2 O 0.1g/L、Na 2 EDTA 10.05g/L、FeCl 3 8.35g/L、CuSO 4 ·5H 2 O 0.16g/L、CoCl 2 ·6H 2 O 0.18g/L.
  9. 9. A fermentation method of recombinant engineering bacteria according to claim 6, wherein the fermentation method comprises the steps of inoculating seed liquid after the activation of the recombinant engineering bacteria into a fermentation medium, and culturing at 37 ℃, wherein the fermentation medium comprises the following components of 10g/L glucose, 10g/L tryptone, 1g/L ammonium citrate, 10g/L xylose, 10g/L nicotinamide, and 3mL/L ampicillin 100µg/mL、K 2 HPO 4 12.54g/L、KH 2 PO 4 2.31g/L、MgSO 4 ·7H 2 O 5g/L、 microelement liquid, and the microelement liquid comprises the following components ：CaCl 2 0.5g/L、ZnSO 4 ·7H 2 O 0.18g/L、MnSO 4 ·H 2 O 0.1g/L、Na 2 -EDTA 10.05g/L、FeCl 3 8.35g/L、CuSO 4 ·5H 2 O 0.16g/L、CoCl 2 ·6H 2 O 0.18g/L.
  10. 10. A method for preparing D-mannitol, comprising the steps of: The fermentation broth obtained by culturing and fermenting the recombinant engineering bacteria according to claim 9, wherein D-fructose and xylose are used as substrates, nicotinamide is used as a reaction medium to form a reaction system, and D-mannitol is obtained after conversion reaction.

Description

Recombinant engineering bacterium and application thereof in preparation of mannitol Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to recombinant engineering bacteria and application thereof in preparing mannitol. Background The enzyme catalytic reaction has the advantages of high selectivity, biodegradability, environmental friendliness and the like. Cofactor-dependent enzymes are critical in the natural product biosynthetic pathway, about 50% of known enzymes are cofactor dependent, often involved in key catalytic steps that are difficult to achieve in organic synthesis, have great potential in the field of biocatalysis, but require a co-factor regeneration system. The dehydrogenase-catalyzed process requires the participation of coenzyme-reduced Nicotinamide Adenine Dinucleotide (NADH) and oxidized nicotinamide adenine dinucleotide (NAD+) or reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and oxidized nicotinamide adenine dinucleotide phosphate (NADP+). The price is very expensive, the property is unstable, the stoichiometric use cost is extremely high, and the method becomes a core bottleneck for industrial application of dehydrogenase catalysis. If it is desired to be used in practical production, it is desirable to greatly reduce the cost of cofactors, including reducing the inhibition of cofactor degradation products, shortening cofactor exposure time, stabilizing cofactors by substrate channeling effects, and the like. D-mannitol is a polyhydric alcohol, widely existing in bacteria, fungi, algae and higher plants, is an important osmotic adjusting substance in organisms, and has an antioxidant function. The current production method of D-mannitol mainly adopts a chemical hydrogenation method, and D-fructose is produced by catalytic hydrogenation of Raney catalyst under high temperature and high pressure. The process for producing D-mannitol by a chemical method has obvious defects, mainly comprising high sorbitol serving as a byproduct and high downstream separation cost. In recent years, the biological synthesis of D-mannitol has been attracting attention, D-mannitol can be produced from D-fructose by the catalysis of D-mannitol dehydrogenase, and the process does not produce sorbitol as a byproduct, but requires the participation of coenzyme NADH. The catalytic process of dehydrogenases must be accompanied by a cycle of coenzymes in practical use. Coenzyme cycling is typically achieved by introducing a second enzyme (co-enzyme) and a second co-substrate (co-substrate) into the system. The coenzyme cycle is achieved by dual enzyme coupling of the target reaction and the regeneration reaction at the expense of the second co-substrate. Under the two common coenzyme circulation systems, even if the second auxiliary enzyme is introduced to circulate the cofactor, a certain amount of cofactor is usually required to be additionally added when the catalytic hydrogenation of the dehydrogenase is actually applied, otherwise, the total amount of the cofactor is too small, the reaction cannot be continued, and the overall conversion rate is low. Disclosure of Invention The invention aims to solve the technical problem of providing the recombinant engineering bacterium and the application thereof in preparing mannitol, wherein the recombinant engineering bacterium is used for catalyzing the reaction of synthesizing D-mannitol from fructose after fermenting to produce enzyme, and no additional cofactor is required to be added in the reaction process, so that the reaction cost can be greatly reduced in actual production, and the recombinant engineering bacterium has a good industrial prospect. The invention provides a recombinant engineering bacterium, which takes escherichia coli as a host, wherein the recombinant engineering bacterium knocks out genes including a gene ptsG encoding protein EIICB Glc in a glucose PTS system, a gene mtlA encoding EIICA protein in D-mannitol transmembrane transport and a gene mtlD of D-mannitol 1-phosphate dehydrogenase of a D-mannitol utilization way, and integrates genes including glucose transport assisting protein glf, glucose kinase glcK, xylose utilization gene cluster xylose isomerase xlyA and xylulophosphorylase xlyB in the genome of the recombinant engineering bacterium, wherein the amino acid sequence of the glucose transport assisting protein glf is shown as SEQ ID NO.1, and the amino acid sequence of the glucose kinase glcK is shown as SEQ ID NO. 2. The invention is realized in this way, and also provides a recombinant engineering bacterium which has been preserved in China center for type culture Collection, with the preservation number of CCTCC NO: M20252967, which is located in China university of Wuhan and Wuhan in 2025, 12 months and 19 days. The classification name of the recombinant engineering bacteria is ESCHERICHIA COLI ECNADMF. Furthermore, the recombinant engineering bacteria are used a