Method for producing L-phenylalanine, 4-hydroxymandelic acid and PHA material through one-carbon resource and application of method

Abstract

The invention provides a method for producing L-phenylalanine, 4-hydroxymandelic acid and / or a PHA material through a one-carbon resource and application of the L-phenylalanine, the 4-hydroxymandelic acid and / or the PHA material. According to the method, a halomonas strain which cannot be directly produced by using a one-carbon resource originally is modified, and a carbon dioxide fixed gene is added, so that recombinant bacteria capable of producing L-phenylalanine and 4-hydroxymandelic acid are constructed, and co-production of extracellular L-phenylalanine, 4-hydroxymandelic acid and other high-added-value compounds and intracellular polyhydroxyalkanoate (PHA) is realized. According to the invention, a more economic, sustainable and high-benefit platform for producing L-phenylalanine, 4-hydroxymandelic acid and PHA is established, and the method plays an important role in further reduction of production cost and improvement of sustainable development.

Description

Technical Field

[0001] The present invention relates to the field of microbial metabolic engineering. More specifically, the present invention relates to a method for producing L-phenylalanine and 4-hydroxymandelic acid using carbon dioxide and its derivatives, etc., and its applications. Background Art

[0002] In recent years, due to the excessive emission of CO 2 , a series of environmental problems such as global warming have occurred. Green development has become an important national strategy. How to reduce the consumption of energy and raw materials and reduce environmental pollution while developing rapidly has become a major problem to be solved currently and in the future. From another perspective, CO 2 is also an abundant, cheap and renewable carbon source. Currently, in the field of microbial fermentation, organic carbon is mainly used as a substrate for fermentation production, and the cost of the carbon source is the main production cost.

[0003] So far, six natural CO 2 fixation pathways have been fully verified. These pathways include the Calvin (Calvin-Benson-Bassham, CBB) cycle, the reductive acetyl-CoA (Wood-Ljungdahl) pathway, the dicarboxylic acid / 4-hydroxybutyric acid (DC / HB) cycle, the 3-hydroxypropionic acid / 4-hydroxybutyric acid (HP / HB) cycle, the 3-hydroxypropionic acid (3-HP) bicyclic cycle, the reductive TCA cycle. In addition, the reductive glycine pathway has also been identified as a potential natural CO 2 fixation pathway. These autotrophic pathways use various enzymes to fix CO 2 or bicarbonate, enabling microorganisms to adapt to different habitats and produce central carbon metabolism intermediates for their growth. However, the typical characteristics of most industrial microorganisms are neutral or acidic fermentation, and the low solubility of CO 2 in the culture limits the practical application of these autotrophic pathways (Liu, Z., Wang, K., Chen, Y., Tan, T., and Nielsen, J. (2020). Third-generation biorefineries as the means to produce fuels and chemicals from CO 2 . Nature Catalysis 3, 274-288).

[0004] Currently, although there are research reports that Escherichia coli uses the CBB cycle to express the exogenous carbon fixation gene ribulose-1,5-bisphosphate carboxylase / oxygenase to achieve CO 2Fixation and growth. However, it can only maintain its own growth, unable to achieve the large-scale production of compounds, and it has a slow growth rate and low biomass (Antonovsky N, Gleizer S, Noor E, Zohar Y, Herz E, Barenholz U, Zelcbuch L, Amram S, Wides A, Tepper N et al (2016). Sugar Synthesis from CO2 in Escherichia coli. Cell, 166(1):115-125.).

[0005] Currently, in industry, the production of L-phenylalanine and 4-hydroxymandelic acid (4HMA) mainly uses chemical synthesis, and microbial synthesis occupies a small market share, mainly concentrated in the production of high-value-added chemicals. In terms of microbial synthesis, recombinant Escherichia coli is mainly used, and glucose and / or xylose are used as substrates for de novo synthesis. Some studies have shown that under the condition of using glucose and xylose as co-substrates, after 60 hours of fermentation in a 5L fermenter, the yield of 4HMA by recombinant Escherichia coli is 15.8 g / L (Li, FF., Zhao, Y., Li, BZ. et al. Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose–xylose mixture. Microb Cell Fact 15, 90 (2016). https: / / doi.org / 10.1186 / s12934-016-0489-4). There are also studies using citric acid and glucose as substrates, and after 76 hours of fermentation in a 5L fermenter, the yield of 4HMA by recombinant Escherichia coli is 32.768 g / L (Liu PP, Jin QW., Li, XY., Zhang RL., Yuan HM., Liu CW., Wang PC., 2024, Directed evolution and metabolic engineering generate an Escherichia coli cell factory for de novo production of 4-hydroxymandelate., Bioresource Technology.), but the fermentation substrates used in these studies are relatively costly. Summary of the Invention

[0006] By knocking out the key glycolysis genes 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate isomerase (pgi) in Halomonas, the present inventors constructed a glycolysis-deficient strain, and then overexpressed the exogenous carbon fixation genes ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), phosphoribulokinase (prk) and glucose-6-phosphate dehydrogenase (gnd) on this basis, achieving the CO 2 -dependent growth and production of Halomonas. Then, through isotope tracer experiments, the present inventors found that most metabolites in the central carbon metabolism of the recombinant strain could be 13 CO 2 -labeled, determining that the recombinant strain could fix carbon dioxide and achieve CO 2 -dependent growth. In addition, the present inventors used non-targeted metabolomics to detect and analyze the cell supernatant after fermentation of the engineered strain, and found that a large amount of phenol, L-phenylalanine and 4-hydroxymandelic acid were produced extracellularly. Through enrichment analysis, it was found that these metabolites all belonged to shikimate pathway metabolites. Secondly, due to the alkaline culture conditions of Halomonas increasing the solubility of CO 2 , the above CO 2 fixation pathway can further promote the high-value application of CO 2 , which has important social and scientific significance for carbon neutrality.

[0007] Therefore, on the one hand, the present invention provides an engineered Halomonas that expresses exogenous ribulose-1,5-bisphosphate carboxylase / oxygenase (Ribulose-1,5-bisphosphate carboxylase / oxygenase, Rubisco) (EC 4.1.1.39), phosphoribulokinase (phosphoribulokinase, PRK) (EC:2.7.1.19) and glucose-6-phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase, GND) (EC 1.1.1.44).

[0008] In a specific embodiment, the engineered Halomonas has knocked out or lacks the endogenous glycolysis key genes 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate isomerase (pgi).

[0009] In a specific embodiment, the Halomonas includes Halomonas bluephagenesis or its derivative, Halomonas campaniensis or its derivative, Halomonas aydingkolgenesis or its derivative, preferably Halomonas bluephagenesis TD01 (deposit number CGMCC No. 4353), Halomonas bluephagenesis TD1.0, Halomonas aydingkolgenesis M1 (deposit number CGMCC No. 19880) or Halomonas campaniensis LS21 (deposit number CGMCC No. 6593). The above strains have all been deposited and have been disclosed in previous patent applications. For example, CGMCC No. 4353, CGMCC No. 19880, and CGMCC No. 6593 have been disclosed in CN119464346A.

[0010] In a specific embodiment, the Rubisco is from Rhodospirillum rubrum, the PRK is from Synechococcus elongatus, and / or the GND is from Leuconostoc lactis; the Rubisco, PRK, and GND are expressed on one or more plasmids or on the genome of the engineered Halomonas (i.e., integrating exogenous Rubisco, PRK, and / or GND into the genome); the Rubisco, PRK, and GND are expressed by a constitutive or inducible promoter, preferably the promoter is the porin promoter, more preferably the porin promoter is porin226; the amino acid sequence of the Rubisco is as shown in SEQ ID NO: 1, the amino acid sequence of the PRK is as shown in SEQ ID NO: 3, and / or the amino acid sequence of the GND is as shown in SEQ ID NO: 5; and / or the gene sequence of the Rubisco is as shown in SEQ ID NO: 2, the gene sequence of the PRK is as shown in SEQ ID NO: 4, and / or the gene sequence of the GND is as shown in SEQ ID NO: 6.

[0011] In a specific embodiment, the constitutive promoter includes a wild-type porin promoter (Pporin) or a mutant thereof, and the mutants include Pporin29, Pporin88, Pporin221, Pporin194, Pporin251, Pporin278, Pporin68, Pporin42, Pporin58, Pporin226, Pporin183, Pporin140 or Pporin141. These porin promoters and their mutants are disclosed in, for example, CN117143793B.

[0012] In a specific embodiment, the inducible promoter includes P tac promoter, P lux promoter or P lac promoter, and the inducers that regulate the inducible promoter include IPTG (isopropyl-β-D-thiogalactoside) or AHL (N-acyl homoserine lactone).

[0013] In a specific embodiment, the Rubisco, PRK and GND encoding genes are operably linked in sequence after the promoter.

[0014] In a specific embodiment, the exogenous Rubisco, PRK and GND encoding genes are inserted into the G4 locus of the Halomonas bluephagenesis genome.

[0015] On the other hand, the present invention provides a method for producing L-phenylalanine and / or 4-hydroxymandelic acid using CO 2 or its derivatives, the method comprising culturing the engineered Halomonas according to the present invention in a medium containing CO 2 or its derivatives. The method is preferably an open fermentation method.

[0016] In a specific embodiment, in the above method, 1) the CO 2 is naturally present in air or is CO 2 produced in industry (such as power plants, cement plants, waste incinerators, steel mills, etc.); and / or 2) the CO 2 derivatives are: i) organic acids and / or organic acid salts with any integer number of C atoms from 1 to 10; and / or, ii) inorganic substances containing CO 3 2- groups and / or HCO 3 - groups, more preferably selected from one or more of carbonates, bicarbonates, formic acid, formates, acetic acid or acetates; even more preferably, the CO 2The derivatives are selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formate or acetate,

[0017] Preferably, the method uses NaHCO 3 , or NaHCO 3 and sodium formate (preferably 14.5 g / L sodium formate and 54 g / L NaHCO 3 , or 29 g / L sodium formate and 36 g / L NaHCO 3 ) combination or NaHCO 3 , CO 2 and sodium formate combination as (carbon source or sole carbon source) substrate to culture the engineered Halomonas.

[0018] In one specific embodiment, 1) the L-phenylalanine and / or 4-hydroxymandelic acid are obtained as extracellular products; 2) the Halomonas can de novo synthesize L-phenylalanine and 4-hydroxymandelic acid through CO 2 or its derivatives; 3) during the process of culturing the engineered Halomonas, the CO 2 or its derivatives as the carbon source of the halophilic bacteria can also adjust the pH value; 4) the culture medium can also include the following substrates: one or more of glucose, gluconate, glycerol, sucrose, xylose or cellulose hydrolysate; 5) the method also includes promoting the production of the L-phenylalanine and 4-hydroxymandelic acid by adjusting the nitrogen source; and / or 6) the engineered Halomonas also produces PHB (intracellular product) and / or phenol (extracellular product).

[0019] On the other hand, the present invention provides a method for fixing carbon dioxide, the method comprising culturing the engineered Halomonas according to the present invention with CO 2 , CO 2 derivatives or a combination thereof as (sole) carbon source. The method is preferably an open fermentation method.

[0020] In one specific embodiment, in the above method, 1) the method fixes carbon dioxide through the Calvin cycle; 2) the CO 2 or CO 2 derivatives are converted into glycerate 3-phosphate together with ribulose 1,5-bisphosphate through the action of ribulose-1,5-bisphosphate carboxylase / oxygenase (EC 4.1.1.39) to achieve the carbon dioxide fixation; 3) the CO 2 is the CO 2 naturally present in the air or generated in industry (power plants, cement plants, waste incineration plants, steel mills, etc.); and / or 4) the CO 2The derivatives are: i) an organic acid and / or an organic acid salt having any integer number of C atoms from 1 to 10; and / or, ii) an inorganic substance containing a CO 3 2- group and / or an HCO 3 - group, more preferably selected from one or more of carbonate, bicarbonate, formic acid, formate, acetic acid or acetate; even more preferably, the CO 2 derivatives are selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formate or acetate, and preferably the method uses NaHCO 3 , or a combination of NaHCO 3 and sodium formate (preferably 14.5 g / L sodium formate and 54 g / L NaHCO 3 , or 29 g / L sodium formate and 36 g / L NaHCO 3 ) or a combination of NaHCO 3 , CO 2 and sodium formate as (a carbon source or the sole carbon source) substrate to culture the engineered Halomonas.

[0021] In a specific embodiment, in the above method, 1) the Halomonas can de novo synthesize L-phenylalanine and / or 4-hydroxymandelic acid through CO 2 or its derivatives; 2) the L-phenylalanine and / or 4-hydroxymandelic acid are obtained as extracellular products; 3) during the culture of the engineered Halomonas, the CO 2 or its derivatives can also regulate the pH value as a carbon source for the halophilic bacteria; 4) the culture medium can also include the following substrates: one or more of glucose, gluconate, glycerol, sucrose, xylose or cellulose hydrolysate; 5) the method also includes promoting the production of L-phenylalanine and 4-hydroxymandelic acid by adjusting the nitrogen source; and / or 6) the engineered Halomonas also produces PHB (intracellular product) and / or phenol (extracellular product). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] From the following detailed description in conjunction with the drawings, the above features and advantages of the present invention will become more apparent, wherein:

[0023] Figure 1 Shows the metabolic engineering modification strategy according to the present invention and the metabolic pathways of 4HMA and L-phenylalanine, with red indicating exogenous genes and blue indicating products. G6P: glucose 6-phosphate; R5P: ribose 5-phosphate; Ru5P: ribulose 1,5-bisphosphate; 3PG: glycerate 3-phosphate; BPG: bisphosphoglycerate; GAP: glyceraldehyde 3-phosphate; E4P: erythrose 4-phosphate; PEP: phosphoenolpyruvate;

[0024] Figure 2 Detection spectra of 4HMA and L-phenylalanine;

[0025] Figure 3 PCR verification spectra for knockout of key glycolysis genes pgi and edd. pgi has two copies, namely pgi1 and pgi2. Primers were designed on both sides of the homologous arms, namely test-pgi1-F, test-pgi1-R; test-pgi2-F, test-pgi2-R; test-edd-F, test-edd-R. These three pairs of primers were used for verification. The knockout band of the pgi1 gene is 1100bp, and that of the wild type TD01 is 2300bp. The knockout band of the pgi2 gene is 1500bp, and that of the wild type TD01 is 3321bp. The knockout band of the edd gene is 1150bp, and that of the wild type TD01 is 2843bp; and

[0026] Figure 4 PCR verification results for carbon fixation pathway knock-in. Primers test-g4-F and test-g4-R were designed on both sides of the homologous arms of the plasmid inserted at G4 for PCR amplification. The size of the gene insertion band is 5200bp, and that of the wild type TD01 is 2100bp. Detailed implementation manners

[0027] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art.

[0028] As used herein, "comprising" or "including" is an open-ended description, including all specified components or steps described, as well as other specified components or steps that do not substantially affect; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0029] As used herein, "and / or" includes all combinations of the items connected by this term, and should be regarded as each combination having been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0030] As used herein, "carbon dioxide fixation" represents the process of converting carbon dioxide or carbon dioxide-derived inorganic salts into organic matter. Of course, it may also include the process of decomposing organic matter that can be converted into carbon dioxide into carbon dioxide and then converting it into organic matter.

[0031] The present invention provides a method for producing L-phenylalanine and 4-hydroxymandelic acid, the method comprising: culturing Halomonas in the presence of CO 2 , CO 2 derivatives or a combination thereof as a substrate to promote the production of the L-phenylalanine and 4-hydroxymandelic acid.

[0032] In a specific embodiment, the Halomonas includes one or more of Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, Halomonas aydingkolgenesis or its derivatives. In a preferred embodiment, the Halomonas is Halomonas bluephagenesis TD1.0 (see Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagenesis TD01 (deposit number CGMCC No. 4353), Halomonas aydingkolgenesis M1 (deposit number CGMCC No. 19880), Halomonas campaniensis LS21 (deposit number CGMCC No. 6593).

[0033] In a specific embodiment, the Halomonas is Halomonas bluephagenesis TD01 (deposit number CGMCC No. 4353).

[0034] In a specific embodiment, the CO 2 is a gas; the CO 2 derivatives are: i) an organic acid and / or an organic acid salt having any integer of 1-10 carbon atoms; and / or, ii) an inorganic substance containing a CO 3 2- root and / or an HCO 3 - root, more preferably selected from one or more of carbonate, bicarbonate, formic acid, formate, acetic acid or acetate; even more preferably, the CO 2 derivatives are selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formate or acetate.

[0035] In a specific embodiment, the substrate includes NaHCO 3 and a combination of CO 2 .

[0036] In a specific embodiment, the Halomonas can grow using the CO 2 or a CO 2 derivative as the (sole) carbon source, wherein the C atoms of the CO 2 or CO 2 derivative are directly converted into the C atoms in one or more of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) through ribulose-1,5-bisphosphate carboxylase / oxygenase in the Calvin cycle. Then it is further converted into L-phenylalanine and 4-hydroxymandelic acid.

[0037] In a specific embodiment, the L-phenylalanine and 4-hydroxymandelic acid are secreted extracellularly. Specifically, the L-phenylalanine and 4-hydroxymandelic acid can be naturally secreted extracellularly by recombinant or engineered Halomonas in an environment enriched with CO 2 , and the environment enriched with CO 2 can be provided by high-concentration CO 2 gas or a CO 2 derivative.

[0038] In a specific embodiment, during the cultivation of recombinant Halomonas, in addition to serving as a carbon source, the CO 2 can also be used to adjust the pH value of the fermentation broth. Specifically, during the cultivation of Halomonas, since the carbonate is continuously consumed as a carbon source, the pH value of the culture medium continuously rises. By directly introducing CO 2 into the culture medium as a carbon source for halophilic bacteria, the pH value of the fermentation broth can be adjusted.

[0039] In a specific embodiment, the method further includes promoting the production of the product by adjusting (such as increasing) the nitrogen source. The nitrogen source includes urea, aspartic acid, glutamine, peptone, yeast extract, etc.

[0040] In a specific embodiment, the substrate further includes one or more of glucose, gluconate, glycerol, sucrose, xylose, or cellulose hydrolysate.

[0041] In a specific embodiment, the CO 2 is the CO 2 naturally present in the air or produced industrially. Under the alkaline culture conditions of halophilic bacteria, any CO 2Gases, including those in the air and those generated industrially, can be used as substrates for the production of aromatic compounds (including phenol, L-phenylalanine, and 4-hydroxymandelic acid).

[0042] In a specific embodiment, the de novo synthesis pathway of the aromatic compound is a process of synthesizing nucleotides using raw materials including phosphoribose, amino acids, one-carbon units, and CO 2 Among them, preferably, the phosphoribose can be produced through the pentose phosphate pathway from one or more of glucose, gluconate, sucrose, xylose, or cellulose hydrolysate; the amino acids include aspartic acid, glutamine, and / or wool hydrolysate; the one-carbon unit can be metabolically produced from one or more of serine, tryptophan, histidine, glycine, methionine, or wool hydrolysate; the CO 2 is provided by CO 2 gas or a CO 2 derivative. Preferably, the C atom of the CO 2 or the CO 2 derivative is directly converted into the C atom of one or more of glyceric acid 3-phosphate (G3P) through ribulose-1,5-bisphosphate carboxylase / oxygenase in the Calvin cycle, and then enters the downstream of glycolysis to be converted into PEP and E4P. More preferably, the process is as Figure 1 shown.

[0043] In a specific embodiment, the one-carbon unit includes one or more of methyl (-CH 3 ), methylene (-CH=), formyl (-CHO), and iminomethyl (-CO=NH).

[0044] In a specific embodiment, the ribulose-1,5-bisphosphate carboxylase / oxygenase belongs to EC4.1.1.39, and preferably its encoding gene is Rubisco. The phosphoribulokinase (PRK) belongs to EC:2.7.1.19, and preferably its encoding gene is prk, and the glucose-6-phosphate dehydrogenase (GND) belongs to EC 1.1.1.44, and preferably its encoding gene is gnd.

[0045] In a specific embodiment, the extracellular products include one or more of L-phenylalanine and 4-hydroxymandelic acid.

[0046] In a specific embodiment, the de novo synthesis pathway of L-phenylalanine and 4-hydroxymandelic acid is the de novo synthesis pathway of aromatic amino acids and 4HMA.

[0047] In a specific embodiment, the de novo synthesis pathway of L-phenylalanine and 4-hydroxymandelic acid is the de novo synthesis pathway of aromatic compounds in Halomonas, and preferably the chassis is Halomonas bluephagenesis TD01.

[0048] In a specific embodiment, the substrate for carbon dioxide fixation includes CO 2 or CO 2 derivatives. Preferably, the CO 2 derivatives are: i) organic acids and / or organic acid salts with any integer number of C atoms from 1 to 10; and / or, ii) inorganic substances containing CO 3 2- radicals and / or HCO 3 - radicals. More preferably, it is selected from one or more of carbonates, bicarbonates, formic acid, formates, acetic acid or acetates; even more preferably, the CO 2 derivatives are selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formates or acetates.

[0049] Those skilled in the art can adjust the addition of CO 2 or CO 2 derivatives according to needs, or maintain the CO 2 and CO 2 in a suitable concentration range during the cultivation process by adding CO 2 derivatives simultaneously.

[0050] CO 2 derivatives are: i) organic acids and / or organic acid salts with any integer number of C atoms from 1 to 10; and / or, ii) inorganic substances containing CO 3 2- radicals and / or HCO 3 - radicals.

[0051] Preferably, the organic acids and / or organic acid salts with any integer number of C atoms from 1 to 10, such as organic acids and / or organic acid salts with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. C atoms. Further preferably, the organic acids and / or organic acid salts are organic acids and / or organic acid salts with reducing power. For example, organic acids and / or organic acid salts that can be decomposed into CO 2 , such as potassium salts, calcium salts, sodium salts, magnesium salts, aluminum salts, zinc salts or iron salts, etc.

[0052] Containing CO 3 2- radicals and / or HCO3 - The inorganic substances in the root include carbonates and / or bicarbonates.

[0053] The cultivation mentioned above includes, but is not limited to, seed cultivation, microplate cultivation, shake flask cultivation, fermenter cultivation, etc.

[0054] The cultivation can be continuous fermentation or fed-batch fermentation.

[0055] Preferably, the substrate is contained in the culture medium.

[0056] The culture medium can be solid, liquid or semi-solid.

[0057] The culture medium can be an activation medium, a seed medium, a fermentation medium, a feeding medium, a supplementary medium, etc.

[0058] Those skilled in the art can adjust the concentrations of the added substrate, nitrogen source and inorganic salts according to the requirements. Among them, the substrate includes a carbon source, CO 2 and / or CO 2 derivatives, the nitrogen source includes urea, aspartic acid, glutamine, peptone, etc., and the inorganic salts maintain the osmotic pressure required by the microorganisms.

[0059] The carbon source includes, but is not limited to, one or more of glucose, gluconate, sucrose, xylose or cellulose hydrolysate. Glucose is preferred.

[0060] In a specific embodiment of the present invention, the substrate includes glucose and / or bicarbonate.

[0061] The cultivation includes, but is not limited to, seed cultivation, shake flask cultivation or fermenter cultivation, etc.

[0062] The cultivation can be continuous fermentation or fed-batch fermentation.

[0063] Preferably, the substrate is contained in the culture medium.

[0064] The culture medium can be solid, liquid or semi-solid.

[0065] The culture medium can be an activation medium, a seed medium, a fermentation medium, a feeding medium, a supplementary medium, etc.

[0066] Preferably, the concentration of the substrate in the culture medium is any value in the range of 1-300 g / L, preferably any value in the range of 30-100 g / L.

[0067] Preferably, the concentration of the carbon source in the culture medium is any value in the range of 1-300 g / L, preferably any value in the range of 30-100 g / L.

[0068] The nitrogen source concentration is any value in the range of 0.001 - 100 g / L.

[0069] Preferably, the concentration of CO 2 and / or CO 2 derivatives in the medium is any value in the range of 1 - 100 g / L, preferably any value in the range of 5 - 40 g / L.

[0070] Preferably, in the medium, the molar amount of CO 2 and / or CO 2 derivatives is any value in the range of 0.01 - 1 M, preferably any value in the range of 0.05 - 1 M, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 M, preferably any value in the range of 0.02 - 0.5 M.

[0071] The carbon source can be added to the medium at the beginning of fermentation, can be fed during fermentation, or can be added as a fed-batch.

[0072] The CO 2 and / or CO 2 derivatives can be added to the medium at the beginning of fermentation, can be fed during fermentation, or can be added as a fed-batch.

[0073] Preferably, the medium further includes inorganic salts, such as potassium salts and / or sodium salts, etc. The potassium salts include but are not limited to one or more of potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium nitrate, or potassium dihydrogen phosphate. The sodium salts include but are not limited to one or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium nitrate, or sodium dihydrogen phosphate. Preferably, the sodium salt is sodium chloride.

[0074] Preferably, in the medium, the concentration of the inorganic salts is any value in the range of 0.0001 - 1 M.

[0075] Wherein, when the CO 2 derivative is an inorganic substance, it can partially replace the inorganic salts (such as NaCl) in the medium.

[0076] Beneficial effects:

[0077] A) The alkaline medium increases the solubility of CO 2 and promotes the sequestration and utilization efficiency of CO 2 B) Adding CO

[0078] / CO 2 / CO 2Derivatives can promote the accumulation of aromatic compounds and broaden the types of high-value products, facilitating the high-value utilization of greenhouse gases. 2

[0079] C) Adding CO 2 / CO 2 Derivatives enable microorganisms to utilize exogenous CO 2 / CO 2 As an additional carbon source, derivatives reduce the use of carbon sources such as glucose, which can lower the cost of industrial production and make the entire production process more green and sustainable.

[0080] The above only summarizes some aspects of the present invention and should not be considered as limiting the present invention in any way.

[0081] All patents and publications mentioned in this application are incorporated into the present invention by reference in their entirety. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention and should not be considered as limiting the present invention or the scope of the specific methods described in the present invention.

[0082] The examples of this application are described by taking the synthesis of L-phenylalanine and 4-hydroxymandelic acid from CO 2 , CO 2 derivatives and / or glucose as an example.

[0083] The experimental methods used in the examples are all conventional methods unless otherwise specified.

[0084] The reagents, materials, etc. used in the examples can be obtained from commercial sources unless otherwise specified.

[0085] Halomonas bluephagenesis TD01 used in the examples is a Gram-negative halophilic bacterium screened from Ayding Lake in Xinjiang by this laboratory (the strain preservation number is CGMCC No. 4353), which has been disclosed in previous patents.

[0086] The gene editing method used in the examples is the CRISPR-Cas9 method. See the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp.[J].Metabolic Engineering,2018,47:219-229.(Qin Q et al.2018)

[0087] The culture medium described in the examples is as follows:

[0088] Table A. Composition of the culture medium​

[0089]

[0090] Method for qualitative and quantitative analysis of aromatic compounds: The cultured product after fermentation was centrifuged at 14,000 g for 5 minutes. 200 μl of the supernatant was taken and 800 μl of anhydrous methanol (pre-cooled in a -80 °C refrigerator) was added. It was incubated at -80 °C for 2 hours. Next, the solution was centrifuged at 4 °C and 14,000 g for 20 minutes, and the supernatant containing metabolites was transferred to a new 1.5 ml test tube and placed on ice. Subsequently, the pellet was dried at room temperature using a Speedvac / freeze dryer, and then the sample was sent to a metabolomics platform for omics determination of cellular metabolites. The relative abundance of aromatic compounds could be determined, and the abundances of the same substance could be compared between groups, but the differences between different substances within a group or between groups could not be compared. At the same time, standard samples of specific substances were sent for detection simultaneously, and the absolute concentrations of 4HMA and phenylalanine could be estimated by the standard curve of the concentration. The specific detection process is as follows: Using a Dionex Ultimate 3000 UPLC system coupled with a TSQ Altis TM Plus triple quadrupole mass spectrometer (Thermo Scientific Fisher, CA) and equipped with a heated electrospray ionization (HESI) probe for metabolite detection. The extract was separated by an ACQUITY UPLC HSS T3 Column (2.1×100 mm, 1.8 μm, Waters). Mobile phase A consisted of 0.01% formic acid and 100% water, and mobile phase B consisted of 0.01% formic acid and 100% acetonitrile. A 12-minute gradient elution was adopted with a flow rate of 300 μL / min, and the gradient conditions were as follows: 0 - 2 minutes, 0% B; 2 - 5 minutes, 0 - 15% B; 5 - 6 minutes, 15 - 95% B; 6 - 7.9 minutes, 95% B; 8 - 12 minutes, 0% B. The column compartment and the sample tray were maintained at 35 °C and 10 °C respectively. The resolutions of the precursor ions and fragment ions were both 0.7 FWHM (full width at half maximum). The ion source parameters were as follows: the positive ion spray voltage was 4500 V; the negative ion spray voltage was 2500 V; the ion transfer tube temperature was 330 °C; the evaporator temperature was 360 °C; the sheath gas flow rate was 45 Arb; the auxiliary gas flow rate was 12 Arb. The collision-induced dissociation (CID) gas pressure was 1.5 mTorr. In the data collected in the selective reaction monitoring (SRM) mode, the precursor ion / product ion of L-phenylalanine was +166.1 / +120, and the precursor ion / product ion of 4-hydroxymandelic acid (4-HMA) was -167 / -121.1. The resolutions of Q1 and Q3 were both 0.7 FWHM.

[0091] Result analysis: Data analysis and quantification were performed using Xcalibur 4.5.445.18 software (Thermo Fisher Scientific) with an external calibration method. The chromatograms of 4HMA and L-phenylalanine are shown in Figure 2 .

[0092] The cell dry weight (CDW, g / L) mentioned in the examples is the ratio of the mass of the dried cells to the volume of the recovered bacterial solution.

[0093] Method for quantifying glucose concentration:

[0094] The remaining glucose concentration in the bacterial cells can be quantified using a liquid chromatograph, an LC-20 instrument (Shimadzu) equipped with an Aminex HPX-87H column (Bio-Rad) and an RID-10A refractive index detector (Shimadzu). The mobile phase is 5 mM degassed H 2 SO 4 , with a flow rate of 0.5 mL / min. The column temperature is maintained at 55 °C. The culture solution is centrifuged at 12,000 × g and 4 °C for 2 min, and the supernatant is taken and filtered through a 0.22 μm polyethersulfone membrane filter (Jinglong) as the sample, with a sample injection volume of 30 μL. Standard products are prepared with glucose at five different concentrations, and a standard curve is plotted to calculate the concentration of the sample.

[0095] The detection method of PHB refers to CN117143793B.

[0096] Example 1: Knocking out the natural glycolysis pathway and constructing an exogenous carbon fixation pathway to construct recombinant Halomonas

[0097] In this experiment, Halomonas bluephagenesis TD01 was used as the starting chassis, and the key glycolysis genes pgi and edd of the strain were knocked out by the CRISPR-Cas9 method. The gRNA sequences for knocking out pgi and edd are shown in Table 1. By aligning with the genomic database, it was found that there are two copies of the pgi gene on the TD genome, namely pgi1 and pgi2, and there is only one copy of the edd gene. After knocking out these three genes using the CRISPR-Cas9 method, primers were designed on both sides of the homologous arms of the knockout plasmid, namely test-pgi1-F, test-pgi1-R; test-pgi2-F, test-pgi2-R; test-edd-F, test-edd-R (the primer sequences are shown in Table 1) for PCR amplification. The knockout band of the pgi1 gene is 1100bp, and that of the wild-type TD01 is 2300bp. The knockout band of the pgi2 gene is 1500bp, and that of the wild-type TD01 is 3321bp. The knockout band of the edd gene is 1150bp, and that of the wild-type TD01 is 2843bp. The PCR verification results of gene knockout of H.bluephagenesis TD01ΔeddΔpgi are shown in Figure 3 . It can be Figure 3 seen that the gene knockout was successful, and the glycolysis-deficient strain H.bluephagenesis TD01ΔeddΔpgi was obtained. Using the sodium chloride medium described above, a microplate experiment was conducted, and three parallel samples were designed in the experiment. The strain H.bluephagenesisTD01ΔeddΔpgi was tested, and the results are shown in Table 2 below. As can be seen from Table 2, in the presence of glucose, the strain H.bluephagenesis TD01ΔeddΔpgi showed growth arrest, while the control group was the wild-type H.bluephagenesisTD01, which showed normal growth.

[0098] Table 1. Detailed information of genes and primers for metabolic engineering modification

[0099]

[0100]

[0101] Table 2. OD of H.bluephagenesis TD01 and H.bluephagenesis TD01.ΔeddΔpgi after growing in glucose medium for 48h 600 Results

[0102] Strain <![CDATA[OD 600 > H.bluephagenesis TD01 1.266±0.0425 H.bluephagenesis TD01.ΔeddΔpgi 0.1417±0.0027

[0103] Based on the glucose-deficient strain, an exogenous carbon fixation pathway (RubRR) was inserted into the genome. This pathway consists of three genes, namely Rubisco, prk, and gnd. The exogenous carbon fixation genes Rubisco from Rhodospirillum rubrum, prk gene from Synechococcus elongatus PCC 7942, and gnd gene from Leuconostoc lactis were selected. The amino acid sequences and gene coding sequences of the three genes are shown in Table 3. Two genomic insertion plasmids carrying the RubRR pathway with different strengths of porin promoters (porin58 and porin226) were constructed (the promoter sequences are shown in Table 1).

[0104]

[0105]

[0106] Using the CRISPR-Cas9 method, the above exogenous carbon fixation RubRR pathway genes were inserted into the G4 locus of the genome of H.bluephagenesis TD01ΔeddΔpgi (the gRNA sequences knocked into the G4 locus are shown in Table 1). Primers test-g4-F and test-g4-R were designed outside the homologous arms to perform PCR verification on the two obtained carbon fixation strains (H.bluephagenesis TD01ΔeddΔpgi RR58 (RR58) and H.bluephagenesis TD01ΔeddΔpgi RR226 (RR226)). The size of the gene insertion band is 5200bp, and that of the wild-type TD01 is 2100bp. The results are shown in Figure 4 . It can be Figure 4 seen that the RubRR pathway genes were successfully inserted.

[0107] The microplate experiment was carried out using two media for cultivation, namely sodium bicarbonate and sodium chloride media. The two strains were tested separately. Three parallels were designed for the experiment, and the results are shown in Table 4 below. It can be seen from Table 4 that in the sodium bicarbonate and sodium chloride media, the strains H.bluephagenesis TD01ΔeddΔpgi and H.bluephagenesis TD01ΔeddΔpgi RR58 showed no growth, the control wild-type H.bluephagenesis TD01 showed normal growth, and the 48h OD of H.bluephagenesis TD01ΔeddΔpgi RR226 600nmSignificantly lower than the control wild type. However, in sodium bicarbonate medium, H.bluephagenesis TD01ΔeddΔpgi RR226 showed growth complementation, and the OD at 48 h 600nm was higher than that of the control group.

[0108] Table 4. OD values of H.bluephagenesis TD01, H.bluephagenesis TD01ΔeddΔpgi and carbon-fixing strains (RR58 and RR226) after 48 h of growth in different media 600 Results

[0109]

[0110] Flask experiments were carried out on H.bluephagenesis TD01, H.bluephagenesis TD01ΔeddΔpgi and H.bluephagenesis TD01ΔeddΔpgi RR226 strains. The results of fermentation at 37 °C and 200 rpm for 48 h using sodium bicarbonate and sodium chloride media are shown in Table 5 below. As can be seen from Table 5, H.bluephagenesis TD01ΔeddΔpgi grew poorly under sodium chloride and sodium bicarbonate culture conditions. Compared with the control, H.bluephagenesis TD01ΔeddΔpgi RR226 had certain growth in sodium chloride and sodium bicarbonate media, but was poorer than the wild-type H.bluephagenesis TD01. This result was consistent with the microplate results. It can be proved that the engineered strain RR226 (H.bluephagenesis TD01ΔeddΔpgi RR226) can utilize sodium bicarbonate for growth.

[0111] Table 5. Biomass results of H.bluephagenesis TD01, H.bluephagenesis TD01ΔeddΔpgi and carbon-fixing strains (R58 and R226) after 48 h of fermentation in different media

[0112]

[0113] Example 2: Exogenous formate addition promotes PHB accumulation in recombinant strains

[0114] Formic acid can be decomposed into CO under the action of formic acid dehydrogenase 2And one molecule of NADH is produced, which can be converted into ATP under the action of the oxidative respiratory chain. Therefore, when performing shake flask fermentation, sodium formate is added to increase the energy within the strain cells. Since the sodium chloride concentration used in previous studies was 50 g / L, with a sodium ion concentration of 0.86 M, while keeping the sodium ion concentration unchanged (maintaining the same osmotic pressure), the corresponding sodium bicarbonate is 72 g / L and sodium formate is 58 g / L. Gradually replace sodium chloride with sodium bicarbonate and sodium formate. Other components of the medium are as described in Table A. Design a shake flask experiment and ferment for 48 h in a constant temperature incubator at 37 °C and 200 rpm. The experiment is designed with three parallel samples. The results are shown in Table 6 below. Compared with the non-added group, in particular, the addition of 14.5 g / L sodium formate and 54 g / L NaHCO 3 promoted the accumulation of PHB in strain RR226 (H.bluephagenesis TD01ΔeddΔpgi RR226).

[0115] Table 6. Results of fermentation of H.bluephagenesis TD01ΔeddΔpgi RR226 in sodium formate medium for 48 h

[0116]

[0117] Example 3: Isotope labeling experiment to verify CO 2 is fixed

[0118] To verify that CO 2 can be fixed as G3P by the carbon fixation gene Rubisco as a substrate, when using strain R226 for fermentation, add 14.5 g / L sodium formate and 54 g / L NaH 13 CO 3 Cultivate using deep microplates. The experiment is designed with three parallel samples. The metabolite results are shown as percentages and the mean value is taken. The culture temperature is 37 °C and the rotation speed is 1000 rpm. The test results are shown in Table 7 below. After adding NaH 13 CO 3 to the medium, most substances in the central carbon metabolism of strain R226 were labeled. Therefore, it can be confirmed that CO 2 can be fixed as G3P by the carbon fixation gene Rubisco as a substrate and participate in the downstream metabolic reactions of glycolysis.

[0119] Table 7. Carbon labeling results of fermentation of H.bluephagenesis TD01ΔeddΔpgi RR226 in NaH 13 CO 3 medium for 48 h

[0120]

[0121]

[0122] Example 4: Fermentation of recombinant Halomonas in a fermenter to determine extracellular product accumulation

[0123] Based on the above research, it has been determined that recombinant Halomonas RR226 can fix CO 2 , participate in the glycolysis reaction, maintain its own growth, and synthesize PHB, but its PHB yield is low. By detecting the residual sugar in the culture medium, it is found that the conversion rate of glucose to PHB of strain R226 is 10.03±2.0563%, which is much lower than the theoretical conversion rate of 48% of wild-type glucose. Therefore, we infer that glucose provides energy to synthesize other metabolites. The R226 strain was amplified and fermented using a 7L fermenter, and the fermenter formula is shown in Table 8 below. At 12, 24, 36, 48, and 60 hours of fermentation, fermentation samples were collected, and the cell precipitate and supernatant were collected by centrifugation. The precipitate was used to calculate the dry cell weight, and the supernatant was fixed with 80% methanol solution for non-targeted metabolomics detection.

[0124] Table 8. Fermentation bottom material formula for 7L fermenter

[0125]

[0126]

[0127] The biomass and PHB yield results of strain RR226 at different time points are shown in Table 9. As can be seen from Table 9, the accumulation of intracellular PHB occurs after 24h. Therefore, the fermentation supernatants collected at 12, 36, 48, and 60h were sent to the metabolomics platform for non-targeted metabolomics detection to obtain a metabolite dataset, and the metabolites were relatively quantified (abundance). Further enrichment analysis found that the abundance of aromatic compounds in the metabolites was relatively high. By analyzing the abundances of phenol, L-phenylalanine, and 4HMA at different time points, it was found that the abundances of these metabolites increased with the increase of fermentation time. The results are shown in Table 10 below. Therefore, it is inferred that through the engineered bacteria constructed by the present invention, CO 2 can be used to produce compounds such as phenol, L-phenylalanine, and 4HMA.

[0128] The absolute quantification of 4HMA and L-phenylalanine in the 60h fermentation supernatant showed that the contents of 4HMA and L-phenylalanine were 144.64mg / L and 1.177mg / L, respectively.

[0129] Table 9. Fermentation results of 7L fermenter

[0130] Fermentation time (h) Biomass (g / L) PHB production (g / L) 12 1.42 0.15 24 2.59 0.24 36 5.44 0.3 48 31.08 7.16 60 39.32 14.49

[0131] Table 10. Abundance of Aromatic Compounds in the Fermentation Broth of 7L Fermenter

[0132] Fermentation time / h 4HMA L-Phenylalanine Phenol 12 2.93E+04 5.07E+05 1.05E+04 36 1.86E+05 7.03E+06 1.05E+04 48 6.08E+07 2.61E+07 1.28E+07 60 8.79E+07 2.81E+07 1.65E+08

[0133] The above results indicate that glucose and CO 2 derivatives are used as common substrates to produce 4HMA and L-phenylalanine as an example. By adding CO 2 derivatives, the accumulation of 4HMA and L-phenylalanine is promoted, and CO 2 is incorporated into high-value chemicals such as 4HMA and L-phenylalanine, which is conducive to the green development of biomanufacturing.

[0134] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0135] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods. In addition, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

1. An engineered Halomonas strain expressing exogenous ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) (EC 4.1.1.39), phosphoribulokinase (PRK) (EC: 2.7.1.19) and glucose-6-phosphate dehydrogenase (GND) (EC 1.1.1.44).

2. The engineered salt mononas according to claim 1, wherein the endogenous glycolysis key genes 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate isomerase (pgi) have been knocked out or deleted.

3. The engineering Halomonas according to claim 1 or 2, wherein the Halomonas comprises Halomonas bluephagenesis or a derivative thereof, Halomonas campaniensis or a derivative thereof, Halomona saydingkolgenesis or a derivative thereof, preferably Halomonas bluephagenesis TD01 (Cultivation Collection Number CGMCC No. 4353), Halomonas bluephagenesis TD1.0, Halomonas aydingkolgenesis M1 (Cultivation Collection Number CGMCC No. 19880) or Halomonas campaniensis LS21 (Cultivation Collection Number CGMCC No. 6593).

4. The engineered Halomonas according to any one of claims 1 to 3, wherein: 1) the Rubisco is from Rhodospirillum rubrum, the PRK is from Synechococcus elongatus, and / or the GND is from Leuconostoc lactis; 2) the Rubisco, PRK and GND are expressed on one or more plasmids or on the genome of the engineered Halomonas; 3) expressing the Rubisco, PRK and GND via a constitutive or inducible promoter, preferably the promoter is a porin promoter, more preferably the porin promoter is porin226; 4) the amino acid sequence of Rubisco is shown in SEQ ID NO: 1, the amino acid sequence of PRK is shown in SEQ ID NO: SEQ ID NO: 3, and / or the amino acid sequence of GND is shown in SEQ ID NO: 5; and / or 5) The gene sequence of Rubisco is shown in SEQ ID NO: 2, the gene sequence of PRK is shown in SEQ ID NO: 4, and / or the gene sequence of GND is shown in SEQ ID NO:

6.

5. A method for producing L-phenylalanine and / or 4-hydroxymandelic acid using CO2 or its derivatives, the method comprising culturing the engineered Halomonas according to any one of claims 1 to 4 in a culture medium containing CO2 or its derivatives.

6. The method according to claim 5, wherein: 1) The CO2 is naturally present in the air or produced in industry (power plants, cement plants, waste incineration plants, steel mills, etc.); and / or 2) The CO2 derivative is: i) an organic acid and / or an organic acid salt having a carbon number of any integer from 1 to 10; and / or, ii) a CO3 2- Roots and / or HCO3 - The inorganic substance of the root is more preferably selected from one or more of carbonate, bicarbonate, formic acid, formate, acetic acid or acetate; more preferably, the CO2 derivative is selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formate or acetate, Preferably, the method utilizes NaHCO3, a combination of NaHCO3 and sodium formate, or a combination of NaHCO3, CO2 and sodium formate as a substrate to culture the engineered Halomonas.

7. The method according to claim 5 or 6, wherein: 1) The L-phenylalanine and / or 4-hydroxymandelic acid are obtained as extracellular products; 2) The Halomonas can synthesize L-phenylalanine and 4-hydroxymandelic acid de novo through CO2 or its derivatives; 3) During the cultivation of the engineered halomonas, the CO2 or its derivatives as a carbon source for the halophilic bacteria can also adjust the pH value; 4) The culture medium may further include the following substrates: One or more of glucose, gluconate, glycerol, sucrose, xylose or cellulose hydrolysate; 5) The method further comprises promoting the production of L-phenylalanine and / or 4-hydroxymandelic acid by adjusting the nitrogen source; and / or 6) The engineered Halomonas also produces PHB and / or phenol.

8. A method for fixing carbon dioxide, the method comprising culturing the engineered Halomonas according to any one of claims 1 to 4 with CO2, a CO2 derivative or a combination thereof as the (sole) carbon source.

9. The method according to claim 8, wherein: 1) The method fixes carbon dioxide through the Calvin cycle; 2) The CO2 or CO2 derivative is converted into triphosphoglycerate together with ribulose 1,5-bisphosphate by the action of ribulose-1,5-bisphosphate carboxylase / oxygenase (EC 4.1.1.39), thereby achieving the carbon dioxide fixation; 3) The CO2 is naturally present in the air or produced in industry (power plants, cement plants, waste incineration plants, steel mills, etc.); and / or 4) The CO2 derivative is: i) an organic acid and / or an organic acid salt having a carbon number of any integer from 1 to 10; and / or, ii) a CO3 2- Roots and / or HCO3 - The inorganic substance of the root is more preferably selected from one or more of carbonate, bicarbonate, formic acid, formate, acetic acid or acetate; more preferably, the CO2 derivative is selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, formate or acetate, Preferably, the method utilizes NaHCO3, a combination of NaHCO3 and sodium formate, or a combination of NaHCO3, CO2 and sodium formate as a substrate to culture the engineered Halomonas.

10. The method according to claim 8 or 9, wherein: 1) The Halomonas bacteria can synthesize L-phenylalanine and / or 4-hydroxymandelic acid de novo by using CO2 or its derivatives; 2) the L-phenylalanine and / or 4-hydroxymandelic acid are obtained as extracellular products; 3) During the cultivation of the engineered halomonas, the CO2 or its derivatives as a carbon source for the halophilic bacteria can also adjust the pH value; 4) The culture medium may further include the following substrates: One or more of glucose, gluconate, glycerol, sucrose, xylose or cellulose hydrolysate; 5) The method further comprises promoting the production of L-phenylalanine and 4-hydroxymandelic acid by adjusting the nitrogen source; and / or 6) The engineered Halomonas also produces PHB and / or phenol.

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