A rothia mucilaginosa engineering strain for producing p-coumaric acid, a construction method and application thereof

Abstract

This invention discloses a chemoautotrophic alkaloid-producing strain of *Alcaligenes rocheborella*, its construction method, and its application. The strain is based on the chemoautotrophic microorganism *Alcaligenes rocheborella* H16 (… Cupriavidus necator Using H16 as the starting strain, the poly-3-hydroxybutyrate (PHB) synthesis pathway of the strain was first knocked out, and then a strain from Rhodotorula glutinis (H16) was introduced. Rhodotorula glutinis ) tyrosine ammonia lyase gene tal A basic engineered strain capable of producing p-coumaric acid was constructed. Subsequently, the throughput of the p-coumaric acid synthesis pathway in the engineered strain was enhanced by overexpressing the endogenous 3-deoxy-7-phosphate heptanulate synthase gene aroG1 and the branching acid synthase gene aroC, and by increasing the tal copy number. Further enhancement was achieved by introducing strains from *Escherichia coli* (…). Escherichia coli By improving the supply of cofactors through the NAD(P) transhydrogenase gene pntAB, a strain of Alcaligenes roximatelye that can synthesize p-coumaric acid using CO2 was finally obtained.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a Roche eugenol-producing alkali-producing strain for producing p-coumaric acid, its construction method, and its application. Background Technology

[0002] Phenylacetic acids are natural compounds, with p-coumaric acid being the most common. It is an important skeletal aromatic compound and a precursor to many high-value products such as caffeic acid and ferulic acid. It has significant value in various fields including medicine, industry, and agriculture. In medicine, p-coumaric acid possesses anticancer, antioxidant, and anti-inflammatory properties and can be used in the preparation of health products and pharmaceuticals. Industrially, it can be used in the preparation of fine cosmetics and fragrances. In agriculture, it can promote plant growth and improve environmental adaptability. The market price for 99% pure p-coumaric acid is around 200,000 RMB per ton, making it expensive. Currently, the main sources of p-coumaric acid are plant extraction and chemical synthesis, methods limited by low yield, low purity, and lack of environmental friendliness.

[0003] Therefore, developing a new biosynthetic method to synthesize p-coumaric acid is of great significance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an engineered strain of *Alcaligenes roximatei* that produces p-coumaric acid, its construction method, and its application, in order to solve the problems existing in the prior art. This invention involves genetic modification of *Alcaligenes roximatei*, resulting in an engineered strain that can fully utilize carbon dioxide to produce p-coumaric acid.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an engineered strain of *Alcaligenes roximatei* that produces p-coumaric acid. This engineered strain is based on *Alcaligenes roximatei* as the starting strain, and after knocking out the phaA, phaB1, and phaC1 genes, it undergoes at least one of the following modifications:

[0007] (1) Transplanting one or more of the foreign genes tal, aro3, aro4, aro7, and pntAB;

[0008] (2) Overexpress one or more of the endogenous genes of the starting strain tktA, tal2346, zwf1, zwf2, zwf3, aroA, aroB, aroC, aroL, aroQ1, aroQ2, aroG1, aroG2, and aroE.

[0009] Alcaligenes rhodotrophogenes is a fast-growing, broad-based, facultative chemoautotrophic microorganism that can grow using carbon dioxide as its sole carbon source. It has been reported that *Alcaligenes rhodotrophogenes* can be used to produce various high-value-added products from carbon dioxide, such as isobutanol as a fuel and humulene as a drug precursor. Therefore, it is inferred that *Alcaligenes rhodotrophogenes* possesses significant potential for the efficient conversion of the greenhouse gas carbon dioxide into coumaric acid.

[0010] Furthermore, the *Alcaligenes roximatei* is *Alcaligenes roximatei* H16.

[0011] Furthermore, the modification of the starting strain includes:

[0012] The exogenous genes tal and pntAB were introduced, and the aroC and aroG1 genes of the starting strain were overexpressed.

[0013] In this invention, the chemoautotrophic microorganism Alcaligenes rhodotrophicus H16 ( Copper-hungry killer Using H16 as the starting strain, the poly-3-hydroxybutyrate (PHB) synthesis pathway of the strain was first knocked out, and then a strain from Rhodotorula glutinis (H16) was introduced. Rhodotorula glutinosa A basic engineered strain capable of producing p-coumaric acid was constructed by overexpressing the endogenous 3-deoxy-7-phosphate heptanulate synthase gene aroG1 and the branching acid synthase gene aroC, and increasing the copy number of tal to enhance the throughput of the p-coumaric acid synthesis pathway in the engineered strain; furthermore, by introducing the tyrosine ammonia lyase gene tal from *Escherichia coli* (E. coli), the throughput was further enhanced. Escherichia coli By improving the supply of cofactors through the NAD(P) transhydrogenase gene pntAB, a strain of Alcaligenes roximatelye that can synthesize p-coumaric acid using CO2 was finally obtained.

[0014] Furthermore, the exogenous gene tal is expressed via an inducible promoter;

[0015] Exogenous genes aro3, aro4, aro7 and endogenous genes tktA, tal2346, aroA, aroB, aroC, aroL, aroQ1, aroQ2, aroG1, aroG2, and aroE are expressed through constitutive or inducible promoters.

[0016] Endogenous genes zwf1, zwf2, and zwf3 and exogenous gene pntAB are expressed via constitutive promoters.

[0017] Furthermore, the exogenous gene tal comes from Rhodotorula glutinis, and its nucleotide sequence is shown in SEQ ID NO. 1;

[0018] The exogenous gene aro3 is from Saccharomyces cerevisiae, and the exogenous genes aro4 and aro7 are both from Yersinia lipolyticis. The corresponding nucleotide sequences are shown in SEQ ID NO. 8-10.

[0019] The exogenous gene pntAB is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO. 5.

[0020] Furthermore, the nucleotide sequence of the endogenous gene zwf1 is shown in SEQ ID NO.2;

[0021] And / or, the nucleotide sequence of the endogenous gene zwf2 is shown in SEQ ID NO.3;

[0022] And / or, the nucleotide sequence of the endogenous gene zwf3 is shown in SEQ ID NO.4;

[0023] And / or, the nucleotide sequence of the endogenous gene tktA is shown in SEQ ID NO.6;

[0024] And / or, the nucleotide sequence of the endogenous gene tal2346 is shown in SEQ ID NO.7;

[0025] And / or, the nucleotide sequence of the endogenous gene aroA is shown in SEQ ID NO.11;

[0026] And / or, the nucleotide sequence of the endogenous gene aroB is shown in SEQ ID NO.12;

[0027] And / or, the nucleotide sequence of the endogenous gene aroC is shown in SEQ ID NO.13;

[0028] And / or, the nucleotide sequence of the endogenous gene aroL is shown in SEQ ID NO.14;

[0029] And / or, the nucleotide sequence of the endogenous gene aroQ1 is shown in SEQ ID NO.15;

[0030] And / or, the nucleotide sequence of the endogenous gene aroQ2 is shown in SEQ ID NO.16;

[0031] And / or, the nucleotide sequence of the endogenous gene aroG1 is shown in SEQ ID NO.17;

[0032] And / or, the nucleotide sequence of the endogenous gene aroG2 is shown in SEQ ID NO.18;

[0033] And / or, the nucleotide sequence of the endogenous gene aroE is shown in SEQ ID NO.19.

[0034] In a second aspect, the present invention provides a method for constructing a *Rochelya eukaryotic* engineered strain for producing p-coumaric acid.

[0035] The construction method includes the following modifications:

[0036] (1) Constructing the lac-PntAB expression cassette;

[0037] (2) Using pUC-TAL and the genome of Alcaligenes roximatei as templates, construct the recombinant plasmid p2MCBAD-TAL-AroG1-AroC-TAL;

[0038] (3) Knock out the phaC1, phaA, and phaB1 genes in Alcaligenes roximatee H16 to construct H16 phaC1AB1;

[0039] (4) Integrate the lac-PntAB expression box into H16 phaC1AB1 to construct H16△phaC1AB1△ldh:lac-PntAB;

[0040] (5) The recombinant plasmid p2MCBAD-TAL-AroG1-AroC-TAL was transferred into H16△phaC1AB1△ldh: lac-PntAB to construct H16△phaC1AB1△ldh: lac-PntAB p2MCBAD-TAL-AroG1-AroC-TAL.

[0041] In this invention, by using yeast derived from red yeast rice ( Rhodotorula glutinosa The tyrosine ammonia-lyase gene tal was synthesized by a gene company (Qingke Biotechnology Co., Ltd.) through codon optimization. The synthesized gene was amplified by PCR and purified and recovered. It was ligated with the amplified linearized plasmid p2MCBAD at a molar ratio of 1:4. The ligation product was transformed into competent E. coli cells, and positive clones were screened on LB solid plates containing chloramphenicol antibiotic. After verification and sequencing, the recombinant plasmid p2MCBAD-TAL was obtained.

[0042] From brewing yeast ( Saccharomyces cerevisiae The gene aro3 and the gene of Yersinia lipolytica ( Yarrowia lipolytica The genes aro4 and aro7 were amplified by PCR, purified, and recovered, and ligated with the linearized plasmid p2MCBAD-TAL at a molar ratio of 1:4. The ligation product was transformed into competent E. coli cells, and positive clones were screened on LB agar plates containing chloramphenicol. After verification and sequencing, the recombinant plasmids p2MCBAD-TAL-Aro3, p2MCBAD-TAL-Aro4, and p2MCBAD-TAL-Aro7 were obtained.

[0043] will come from C. necator The endogenous genes of H16, tktA, tal2346, aroA, aroB, aroC, aroL, aroQ1, aroQ2, aroG1, aroG2, aroE, and self-adhesive red yeast ( Rhodotorula glutinosa The tyrosine ammonia-lyase gene tal, purified and recovered by PCR amplification, was ligated with the linearized plasmid p2MCBAD-TAL at a molar ratio of 1:4. The ligation product was transformed into competent E. coli cells and cultured on LB lysate containing chloramphenicol. Positive clones were screened on solid plates, and after verification and sequencing, recombinant plasmids p2MCBAD-TAL-TktA, p2MCBAD-TAL-Tal2346, p2MCBAD-TAL-AroA, p2MCBAD-TAL-AroB, p2MCBAD-TAL-AroC, p2MCBAD-TAL-AroL, p2MCBAD-TAL-AroQ1, p2MCBAD-TAL-AroQ2, p2MCBAD-TAL-AroG1, p2MCBAD-TAL-AroG2, p2MCBAD-TAL-AroE, p2MCBAD-TAL-AroG1-AroC, and p2MCBAD-TAL-AroG1-AroC-TAL were obtained.

[0044] Escherichia coli ( Escherichia coli The pntAB gene and from C. necator The endogenous genes zwf1, zwf2, and zwf3 of H16, along with the purified and linearized plasmid pK18mobSacB-ldhud-lac obtained after PCR amplification, were ligated at a molar ratio of 1:4. The ligation product was transformed into competent E. coli cells, and positive clones were screened on LB agar plates containing chloramphenicol. After verification and sequencing, recombinant plasmids pK18mobSacB-ldhud-lac-PntAB, pK18mobSacB-ldhud-lac-Zwf1, pK18mobSacB-ldhud-lac-Zwf2, and pK18mobSacB-ldhud-lac-Zwf3 were obtained. Subsequently, the expression cassettes lac-PntAB, lac-Zwf1, lac-Zwf2, and lac-Zwf3 were integrated into the genome of the engineered strain through genome integration.

[0045] Escherichia coli DH5α containing the recombinant plasmid was used as the donor bacterium, and Escherichia coli T1 containing the pRK2013 plasmid was used as the helper bacterium. C. necator H16 gene deletion strain H16△ phaC 1 FROM 1The recipient bacteria were bound and the positive recombinant strain was obtained by PCR verification.

[0046] In a third aspect, the present invention provides the application of any of the above-described engineered strains of *Rochelys eutrophicus* in the production of p-coumaric acid.

[0047] The present invention also provides a method for producing p-coumaric acid, wherein p-coumaric acid is produced by sealed fermentation culture using any of the above-described engineered strains of *Rochelys eutrophicus*; and a mixed gas containing CO2 is introduced during fermentation culture.

[0048] Furthermore, the mixed gas includes CO2, H2 and O2, and the volume mixing ratio of CO2, H2 and O2 is 1:6~8:2~3.

[0049] In the microbial synthesis of p-coumaric acid, the naturally occurring shikimic acid pathway in microorganisms was utilized. The synthesis of p-coumaric acid from CO2 was first achieved by introducing the exogenous gene *tal*. Based on this, the shikimic acid pathway was further optimized to increase p-coumaric acid yield by introducing exogenous genes *aro3*, *aro4*, *aro7*, and *pntAB*, as well as overexpressing endogenous key genes *tktA*, *tal2346*, *zwf1*, *zwf2*, *zwf3*, *aroA*, *aroB*, *aroC*, *aroL*, *aroQ1*, *aroQ2*, *aroG1*, *aroG2*, and *aroE*.

[0050] This invention uses p2MCBAD as the base plasmid and connects it to the codon-optimized tyrosine ammonia-lyase gene tal, which can catalyze the conversion of substrates to coumaric acid, to construct the vector p2MCBAD-TAL. The resulting recombinant plasmid is then transformed into... phaC 1 FROM 1 Gene-deleted Alcaligenes roximatee H16 (H16∆) phaC 1 FROM 1 A recombinant strain capable of producing p-coumaric acid was obtained. During fermentation, 0.1 g / L arabinose was used for induction. The fermentation broth was tested after 120 hours of induction. The coumaric acid yield was the highest and tended to stabilize, with an optimal yield of 15.4 mg / L. Further optimization of the arabinose induction concentration and the expression intensity of the tyrosine ammonia-lyase gene further increased the yield to 18.7 mg / L.

[0051] This invention uses p2MCBAD-TAL as the base plasmid and overexpresses the endogenous 3-deoxy-7-phosphate heptanulate synthase gene aroG1 and the branching acid synthase gene aroC in the shikimic acid pathway to increase the copy number of the tyrosine ammonia-lyase gene tal, constructing the vector p2MCBAD-TAL-AroG1-AroC-TAL. This recombinant plasmid is then transformed into H16△phaC1AB1, which integrates the lac-PntAB expression cassette, ultimately obtaining the engineered strain H16△. phaC 1 FROM 1 △ldh: lac-PntAB p2MCBAD-TAL-AroG1-AroC-TAL. After continuous culture with a constant supply of CO2 for 168 hours, this strain yielded 62.2 mg / L of p-coumaric acid.

[0052] The beneficial effects of this invention include at least the following:

[0053] This invention provides a *Alcaligenes roximatei* engineered bacterium capable of producing p-coumaric acid using carbon dioxide. This method offers advantages such as high yield, low cost, and being environmentally friendly and pollution-free, making it suitable for the commercial production of p-coumaric acid. Attached Figure Description

[0054] Figure 1 The plasmid map of the constructed vector p2MCBAD-TAL.

[0055] Figure 2 The plasmid map of the constructed vector pK18mobSacB-ldhud-lac-PntAB.

[0056] Figure 3 The plasmid map of the constructed vector pK18mobSacB-ldhud-lac-Zwf1.

[0057] Figure 4 The plasmid map of the constructed vector pK18mobSacB-ldhud-lac-Zwf2.

[0058] Figure 5 The plasmid map of the constructed vector pK18mobSacB-ldhud-lac-Zwf3.

[0059] Figure 6 The plasmid map of the constructed vector p2MCBAD-TAL-TktA.

[0060] Figure 7 The plasmid map of the constructed vector p2MCBAD-TAL-Tal2346.

[0061] Figure 8 The plasmid map of the constructed vector p2MCBAD-TAL-Aro3.

[0062] Figure 9 The plasmid map of the constructed vector p2MCBAD-TAL-Aro4.

[0063] Figure 10 The plasmid map of the constructed vector p2MCBAD-TAL-Aro7.

[0064] Figure 11 The plasmid map of the constructed vector p2MCBAD-TAL-AroA.

[0065] Figure 12 The plasmid map of the constructed vector p2MCBAD-TAL-AroB.

[0066] Figure 13 The plasmid map of the constructed vector p2MCBAD-TAL-AroC.

[0067] Figure 14 The plasmid map of the constructed vector p2MCBAD-TAL-AroL.

[0068] Figure 15 The plasmid map of the constructed vector p2MCBAD-TAL-AroQ1.

[0069] Figure 16 The plasmid map of the constructed vector p2MCBAD-TAL-AroQ2.

[0070] Figure 17 The plasmid map of the constructed vector p2MCBAD-TAL-AroG1.

[0071] Figure 18 The plasmid map of the constructed vector p2MCBAD-TAL-AroG2.

[0072] Figure 19 The plasmid map of the constructed vector p2MCBAD-TAL-AroE.

[0073] Figure 20 The plasmid map of the constructed vector p2MCBAD-TAL-AroG1-AroC.

[0074] Figure 21 The plasmid map of the constructed vector p2MCBAD-TAL-AroG1-AroC-TAL.

[0075] Figure 22 The figure shows the fermentation results of the optimized recombinant strain for coumaric acid.

[0076] Figure 23 The figure shows the fermentation results of the recombinant strain with the optimized induction concentration.

[0077] Figure 24 The fermentation results of the recombinant strain that enhanced the shikimic acid pathway are shown in the figure.

[0078] Figure 25 The fermentation results of the optimized cofactor recombinant strain are shown in the figure. Detailed Implementation

[0079] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0080] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0081] The following specific embodiments illustrate the solution proposed in this invention:

[0082] Example 1: Basic Strains and Reagents

[0083] Table 1. Strains and plasmids

[0084]

[0085] Table 2 Experimental Reagents

[0086]

[0087] Example 2: Gene Acquisition and Vector Construction

[0088] The source of red yeast ( Rhodotorula glutinosa )of such Its nucleotide sequence is shown in SEQ ID No. 1. It was chemically synthesized by BGI Genomics and inserted into the pUC-57 vector to obtain the pUC-TAL vector.

[0089] Will come from ( Copper-hungry killer H16) zwf1 The gene, whose nucleotide sequence is shown in SEQ ID No. 2, is composed of... Copper-hungry killer H16 genome amplification was used to obtain it.

[0090] Will come from ( Copper-hungry killer H16) zwf2 The gene, whose nucleotide sequence is shown in SEQ ID No. 3, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0091] Will come from ( Copper-hungry killer H16) zwf3 The gene, whose nucleotide sequence is shown in SEQ ID No. 4, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0092] Will originate from E. coli ( Escherichia coli) of pntAB The gene, whose nucleotide sequence is shown in SEQ ID No. 5, was chemically synthesized by BGI Genomics and inserted into the pUC-57 vector to obtain the pUC-PntAB vector.

[0093] Will come from ( Copper-hungry killer H16) tktA The gene, whose nucleotide sequence is shown in SEQ ID No. 6, is composed of... Copper-hungry killer H16 genome amplification obtained

[0094] Source ( Copper-seeking killer H16 )of tal2346 The gene, whose nucleotide sequence is shown in SEQ ID No. 7, is composed of... Copper-hungry killer H16 genome amplification obtained

[0095] From brewer's yeast ( Saccharomyces cerevisiae )of aro3 The gene, whose nucleotide sequence is shown in SEQ ID No. 8, was chemically synthesized by BGI Genomics and inserted into the pUC-57 vector to obtain the pUC-Aro3 vector.

[0096] The yeast derived from Yersinia lipolytica ( Yarrowia lipolytica )of aro4 The gene, whose nucleotide sequence is shown in SEQ ID No. 9, was chemically synthesized by BGI Genomics and inserted into the pUC-57 vector to obtain the pUC-Aro4 vector.

[0097] The yeast derived from Yersinia lipolytica ( Yarrowia lipolytica )of aro7 The gene, whose nucleotide sequence is shown in SEQ ID No. 10, was chemically synthesized by BGI Genomics and inserted into the pUC-57 vector to obtain the pUC-Aro7 vector.

[0098] Will come from ( Copper-hungry killer H16) aroA The gene, whose nucleotide sequence is shown in SEQ ID No. 11, is composed of... Copper-hungry killerH16 genome amplification was obtained.

[0099] Will come from ( Copper-hungry killer H16) aroB The gene, whose nucleotide sequence is shown in SEQ ID No. 12, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0100] Will come from ( Copper-hungry killer H16) aroC The gene, whose nucleotide sequence is shown in SEQ ID No. 13, is composed of... Copper-hungry killer H16 genome amplification was used to obtain it.

[0101] Will come from ( Copper-hungry killer H16) aroL The gene, whose nucleotide sequence is shown in SEQ ID No. 14, is composed of... Copper-hungry killer H16 genome amplification was used to obtain it.

[0102] Will come from ( Copper-hungry killer H16) aroQ1 The gene, whose nucleotide sequence is shown in SEQ ID No. 15, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0103] Will come from ( Copper-hungry killer H16) aroQ2 The gene, whose nucleotide sequence is shown in SEQ ID No. 16, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0104] Will come from ( Copper-hungry killer H16) aroG1 The gene, whose nucleotide sequence is shown in SEQ ID No. 17, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0105] Will come from ( Copper-hungry killer H16) aroG2 The gene, whose nucleotide sequence is shown in SEQ ID No. 18, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0106] Will come from ( Copper-hungry killer H16) aroE The gene, whose nucleotide sequence is shown in SEQ ID No. 19, is composed of... Copper-hungry killer H16 genome amplification was obtained.

[0107] In this embodiment of the invention, the such The nucleotide sequence is shown in SEQ ID NO. 1:

[0108] In this embodiment of the invention, the zwf1 The nucleotide sequence is shown in SEQ ID NO.2:

[0109] In this embodiment of the invention, the zwf2 The nucleotide sequence is shown in SEQ ID NO.3:

[0110] In this embodiment of the invention, the zwf3 The nucleotide sequence is shown in SEQ ID NO. 4:

[0111] In this embodiment of the invention, the pntAB The nucleotide sequence is shown in SEQ ID NO.5:

[0112] In this embodiment of the invention, the tktA The nucleotide sequence is shown in SEQ ID NO.6:

[0113] In this embodiment of the invention, the tal2346 The nucleotide sequence is shown in SEQ ID NO.7:

[0114] In this embodiment of the invention, the aro3 The nucleotide sequence is shown in SEQ ID NO.8:

[0115] In this embodiment of the invention, the aro4 The nucleotide sequence is shown in SEQ ID NO. 9:

[0116] In this embodiment of the invention, the aro7 The nucleotide sequence is shown in SEQ ID NO.10:

[0117] In this embodiment of the invention, the aroA The nucleotide sequence is shown in SEQ ID NO. 11:

[0118] In this embodiment of the invention, the aroB The nucleotide sequence is shown in SEQ ID NO.12:

[0119] In this embodiment of the invention, the aroC The nucleotide sequence is shown in SEQ ID NO.13:

[0120] In this embodiment of the invention, the aroL The nucleotide sequence is shown in SEQ ID NO.14:

[0121] In this embodiment of the invention, the aroQ1 The nucleotide sequence is shown in SEQ ID NO. 15:

[0122] In this embodiment of the invention, the aroQ2 The nucleotide sequence is shown in SEQ ID NO.16:

[0123] In this embodiment of the invention, the aroG1 The nucleotide sequence is shown in SEQ ID NO.17:

[0124] In this embodiment of the invention, the aroG2 The nucleotide sequence is shown in SEQ ID NO. 18:

[0125] In this embodiment of the invention, the aroE The nucleotide sequence is shown in SEQ ID NO.19:

[0126] 2. Construction of the p2MCBAD-TAL vector

[0127] Using pUC-TAL as a template, primers TAL-F and TAL-R were used to perform polymerase chain reaction (PCR) to amplify... tal The PCR amplification system for the gene fragment is shown in the table below:

[0128]

[0129] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 62ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 6ºC to infinity.

[0130] The primer sequences are shown below:

[0131]

[0132] PCR products were purified using a gel extraction and purification kit (SparkJade, catalog number AE0101-C).

[0133] Using the commercial vector pBAD (purchased from Jixiang Baina Biotechnology (Tianjin) Co., Ltd.) as a template, and primers BAD-RF and BAD-RR, polymerase chain reaction (PCR) was performed to expand the BAD promoter fragment. The PCR expansion system is shown in the table below:

[0134]

[0135] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 52ºC for 15 s, 72ºC for 5 min); 72ºC for 5 min; 6ºC to infinity.

[0136] The primer sequences are shown in the table below:

[0137]

[0138] PCR products were purified using a gel extraction and purification kit (SparkJade, catalog number AE0101-C).

[0139] Using the commercial vector pBBR1MCS1 (purchased from Jixiang Baina Biotechnology (Tianjin) Co., Ltd.) as a template, and primers p2MC RF and p2MC RR as primers, polymerase chain reaction (PCR) was performed to amplify the p2MC plasmid framework. The PCR amplification system is shown in the table below:

[0140]

[0141] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 58ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 6ºC to infinity.

[0142] The primer sequences are shown in SEQ ID No. 24 and SEQ ID No. 25:

[0143]

[0144] PCR products were purified using a gel extraction and purification kit (SparkJade, catalog number AE0101-C).

[0145] The TAL fragment and BAD promoter fragment were linked to the linearized vector p2MC as a framework using seamless cloning. The molar ratio of the TAL fragment, BAD promoter fragment to the p2MC plasmid framework was 1:2:3. The mixture was added to a 200 μL EP tube to form a 10 μL system, as shown below:

[0146]

[0147] The ligation system was incubated at 50ºC for 45 min. The ligation product was then converted. E. coli DH5α competent cells were plated onto LB agar plates containing 30 mg / L chloramphenicol. Positive clones were screened by PCR, and recombinant plasmid p2MCBAD-TAL was extracted from the positive clones. Figure 1 Then, it is identified by restriction enzyme digestion and sequencing.

[0148] 3. Construction of vectors enhancing p2MCBAD-TAL-Aro3, p2MCBAD-TAL-Aro4, p2MCBAD-TAL-Aro7, p2MCBAD-TAL-TktA, p2MCBAD-TAL-Tal2346, p2MCBAD-TAL-AroA, p2MCBAD-TAL-AroB, p2MCBAD-TAL-AroC, p2MCBAD-TAL-AroL, p2MCBAD-TAL-AroQ1, p2MCBAD-TAL-AroQ2, p2MCBAD-TAL-AroG1, p2MCBAD-TAL-AroG2, and p2MCBAD-TAL-AroE.

[0149] Using pUC-Aro3, pUC-Aro4, and pUC-Aro7 as templates, primers were Aro3-F, Aro3-R, Aro4-F, Aro4-R, and Aro7-F, Aro7-R, respectively. Using the genome of *Alcaligenes roximatei* as a template, primers were TktA-F, TktA-R, Tal2346-F, Tal2346-R, AroA-F, and AroA-R, respectively. The following fragments were amplified using polymerase chain reaction (PCR) with the following amplification systems: oB-F, AROB-R, AroC-F, AroC-R, AroL-F, AroL-R, AroQ1-F, AroQ1-R, AroQ2-F, AROQ2-R, AroG1-F, AroG1-R, AroG2-F, AroG2-R, and AroE-F, AroE-R. The PCR amplification system is shown below:

[0150]

[0151] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 68ºC for 15 s, 72ºC for 1 min 30 s); 72ºC for 5 min; 6ºC for ∞.

[0152] The primer sequences are shown below:

[0153]

[0154] Using p2MCBAD-TAL as a template, and primers p2MCBAD-TAL-RF and p2MCBAD-TAL-RR, polymerase chain reaction (PCR) was performed to amplify the p2MCBAD-TAL plasmid framework. The PCR amplification system is shown in the table below:

[0155]

[0156] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 58ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 6ºC to infinity.

[0157] The primer sequences are shown in SEQ ID No. 54 and SEQ ID No. 55:

[0158]

[0159] The amplified fragment was ligated to the p2MCBAD-TAL plasmid framework using seamless cloning. The molar ratio of the amplified fragment to the p2MCBAD-TAL plasmid framework was 1:4, and the system is shown below:

[0160]

[0161] The ligation system was incubated at 50ºC for 30 min. The ligation product was then converted. E. coli DH5α competent cells were plated onto LB agar plates containing 34 mg / L chloramphenicol. Positive clones were screened by PCR, and recombinant plasmids p2MCBAD-TAL-Aro3, p2MCBAD-TAL-Aro4, p2MCBAD-TAL-Aro7, p2MCBAD-TAL-TktA, p2MCBAD-TAL-Tal2346, p2MCBAD-TAL-AroA, p2MCBAD-TAL-AroB, p2MCBAD-TAL-AroC, p2MCBAD-TAL-AroL, p2MCBAD-TAL-AroQ1, p2MCBAD-TAL-AroQ2, p2MCBAD-TAL-AroG1, p2MCBAD-TAL-AroG2, and p2MCBAD-TAL-AroE were extracted from the positive clones. 。

[0162] 4. Construction of cofactor enhancement expression cassette integration vectors pK18mobSacB-ldhud-lac-PntAB, pK18mobSacB-ldhud-lac-Zwf1, pK18mobSacB-ldhud-lac-Zwf2, and pK18mobSacB-ldhud-lac-Zwf3

[0163] Polymerase chain reaction (PCR) was performed using the genome of *Alcaligenes roximatei* as a template with primers Zwf1-F, Zwf1-R, Zwf2-F, Zwf2-R, Zwf3-F, Zwf3-R, Ldhup-F, Ldhup-R, Ldhdown-F, and Ldhdown-R; pUC-PntAB as a template with primers PntAB-F and PntAB-R; and the commercial vector pBBR1MCS2 (purchased from Jixiang Baina Biotechnology (Tianjin) Co., Ltd.) as a template with primers lac-F and lac-R to amplify the fragments. The PCR amplification systems are shown below:

[0164]

[0165] The PCR program was: 95ºC for 3 min; 30 cycles × (95ºC 15 s, 68ºC 15 s, 72ºC 1 min 30 s); 72ºC for 5 min; 6ºC to infinity. The primer sequences are shown below:

[0166]

[0167] Using pK18mobSacB as a template, and primers pK18mobSacB-RF and pK18mobSacB-RR, polymerase chain reaction (PCR) was performed to amplify the pK18mobSacB vector framework. The PCR amplification system is shown in the table below:

[0168]

[0169] The PCR program was: 95ºC for 3 min; 30 cycles × (95ºC 15 s, 68ºC 15 s, 72ºC 1 min 30 s); 72ºC for 5 min; 6ºC to infinity. The primer sequences are shown below:

[0170]

[0171] The amplified fragment was ligated to the pK18mobSacB framework using seamless cloning. The molar ratio of the amplified fragment to the pK18mobSacB plasmid was 1:4, as shown in the following system:

[0172]

[0173] The ligation system was incubated at 50ºC for 30 min. The ligation product was then converted. E. coliDH5α competent cells were plated onto LB agar plates containing 34 mg / L chloramphenicol. Positive clones were screened by PCR, and recombinant plasmids pK18mobSacB-ldhud-lac-PntAB, pK18mobSacB-ldhud-lac-Zwf1, pK18mobSacB-ldhud-lac-Zwf2, and pK18mobSacB-ldhud-lac-Zwf3 were extracted from the positive clones. 。

[0174] 5. Construction of effective gene integration vectors p2MCBAD-TAL-AroG1-AroC and p2MCBAD-TAL-AroG1-AroC-TAL

[0175] (1) Construction of p2MCBAD-TAL-AroG1-AroC vector

[0176] Using the genome of *Alcaligenes roximatei* as a template, polymerase chain reaction (PCR) was performed using primers ZHAroG1-F, ZHRAroG1-R, ZHAroC-F, and ZHRAroC-R to amplify the fragment. The PCR amplification system is shown below:

[0177]

[0178] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 68ºC for 15 s, 72ºC for 1 min 30 s); 72ºC for 5 min; 16ºC to infinity.

[0179] The primer sequences are shown below:

[0180]

[0181] Using p2MCBAD-TAL as a template, and primers p2MCBAD-TAL-RF and p2MCBAD-TAL-RR, polymerase chain reaction (PCR) was performed to amplify the p2MCBAD-TAL plasmid framework. The PCR amplification system is shown in the table below:

[0182]

[0183] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 58ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 6ºC to infinity.

[0184] The primer sequences are shown below:

[0185]

[0186] The amplified fragment was linked to the p2MCBAD-TAL plasmid framework using seamless cloning. The molar ratio of the amplified fragment to the p2MCBAD-TAL plasmid framework was 1:4, as shown in the following system:

[0187]

[0188] The ligation system was incubated at 50ºC for 30 min. The ligation product was then converted. E. coli DH5α competent cells were plated onto LB agar plates containing 34 mg / L chloramphenicol, and positive clones were screened by PCR. The recombinant plasmid p2MCBAD-TAL-AroG1-AroC was extracted from the positive clones.

[0189] (2) Construction of p2MCBAD-TAL-AroG1-AroC-TAL expression vector

[0190] Using pUC-TAL as a template, primers TAL2-F and TAL2-R were used. Using the genome of *Alcaligenes roximatei* as a template, primers ZHAroG1-F, ZHRAroG1-R, ZHAroC-F, and ZHRAroC-R2 were used to amplify the fragment via polymerase chain reaction (PCR). The PCR amplification system is shown below:

[0191]

[0192] The PCR program was: 95ºC for 3 min; 30 cycles × (95ºC 15 s, 68ºC 15 s, 72ºC 1 min 30 s); 72ºC for 5 min; 16ºC to infinity. The primer sequences are shown below:

[0193]

[0194] Using p2MCBAD-TAL as a template, and primers p2MCBAD-TAL-RF and p2MCBAD-TAL-RR, polymerase chain reaction (PCR) was performed to amplify the p2MCBAD-TAL plasmid framework. The PCR amplification system is shown in the table below:

[0195]

[0196] The PCR program was as follows: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 58ºC for 15 s, 72ºC for 2 min); 72ºC for 5 min; 6ºC to infinity.

[0197] The primer sequences are shown below:

[0198]

[0199] The amplified fragment was ligated to the p2MCBAD-TAL plasmid framework using seamless cloning. The molar ratio of the amplified fragment to the p2MCBAD-TAL plasmid framework was 1:4, and the system is shown below:

[0200]

[0201] The ligation system was incubated at 50ºC for 30 min. The ligation product was then converted. E. coli DH5α competent cells were plated onto LB agar plates containing 34 mg / L chloramphenicol, and positive clones were screened by PCR. The recombinant plasmid p2MCBAD-TAL-AroG1-AroC-TAL was extracted from the positive clones.

[0202] Example 3

[0203] 1. Construction and identification of gene expression engineered strains

[0204] (1) Strains preparation: containing the construction vector E. coli As a donor bacterium, it contains the helper plasmid pRK2013. E. coli As auxiliary bacteria, C. necator H16 or gene-edited strains were used as recipient bacteria.

[0205] (2) Triparental union

[0206] ① Collect bacterial cells: Collect 2 ml of donor bacteria, 2 ml of helper bacteria, and 2 ml of recipient bacteria, centrifuge at 4000 rpm for 10 min, and discard the supernatant.

[0207] ② Rinse the bacterial cells: Add 500 μl of LB liquid medium to the remaining bacterial cells to resuspend the cells, centrifuge at 4000 rpm for 10 min, and discard the supernatant.

[0208] ③ Resuspension titration: Add 100 μl of LB liquid medium to the remaining bacterial cells to resuspend the cells, and titrate it onto the center of antibiotic-free LB agar plate filter paper. Let it stand in a clean bench for 1 h. Then incubate at 30 ℃ for 12-14 h.

[0209] ④ Spreading on plates: Scrape the bacterial growth into 1 ml of sterile water and mix by blowing and aspiration. Take 100 μl and spread it on a double-antibody (CM, GM) plate. Incubate at 30 ℃ for 48-72 h. The growing strain is the H16 strain carrying the vector.

[0210] ⑤ Transfer to liquid medium: Transfer the bacteria on the double-antibody plate to LB liquid medium, add double antibodies (CM, GM), and incubate at 30 ℃ and 220 rpm for 48-72 h.

[0211] ⑥ After liquid growth, bacterial culture verification was performed using primers BAD YF / ZC YR (sequences below) for colony PCR to verify the recombinant strain. After successful verification, the bacteria were preserved in glycerol tubes, completing the construction of the engineered bacteria.

[0212]

[0213] 2. Construction and identification of genome-integrating strains

[0214] (1) Strains preparation: containing integration vectors E. coli S17-1 was used as the donor bacteria. C. necator H16 or gene-edited strains were used as recipient bacteria.

[0215] (2) Parental union

[0216] ① Collect bacterial cells: Collect 10 ml of donor bacteria and 5 ml of recipient bacteria, centrifuge at 4000 rpm for 10 min, and discard the supernatant.

[0217] ② Rinse the bacterial cells: Add 1 ml of LB liquid medium to the remaining bacterial cells to resuspend the cells, centrifuge at 4000 rpm for 10 min, and discard the supernatant.

[0218] ③ Resuspension titration: Add 100 μl of LB liquid medium to the remaining bacterial cells to resuspend the cells, and titrate it onto the center of antibiotic-free LB agar plate filter paper. Let it stand in a clean bench for 1 h. Then incubate at 30 ℃ for 12-14 h.

[0219] ④ Spreading on plates: Scrape the bacterial growth into 1 ml of sterile water and mix by blowing and aspiration. Take 100 μl and spread it on a double-antibody (CM, GM) plate. Incubate at 30 ℃ for 48-72 h. The growing strain is the H16 strain carrying the vector.

[0220] ⑤ Transfer to liquid medium: Transfer the bacteria on the double-antibody plate to LB liquid medium, add double antibodies (CM, GM), and incubate at 30 ℃ and 220 rpm for 48-72 h.

[0221] ⑥ Sucrose plate preparation: The obtained culture was inoculated into LB medium supplemented with Gm but without sodium chloride (NaCl). After 72 h of incubation, the resulting culture was inoculated onto specific LB plates supplemented with Gm but without NaCl, with an additional 20% sucrose added.

[0222] ⑥ Extract the genome from the cultured strains and use the primers LdhUP F / LdhDOWN R (sequences below) to perform colony PCR to verify the recombinant strains.

[0223]

[0224] Example 4

[0225] 1. Preparation of strain seed culture

[0226] The correctly sequenced engineered bacterial strain was transferred to MSM liquid medium containing 50 mg / L chloramphenicol and 40 mg / L gentamicin sulfate and cultured at 30°C. Five mL of the overnight activated bacterial culture was transferred to 45 mL of MSM liquid medium, to which chloramphenicol, gentamicin sulfate, and fructose were added to a final concentration of 50 mg / L, 40 mg / L, and 16.0 g / L, respectively. Once the bacterial culture reached the logarithmic growth phase, it was used as a seed culture for autotrophic culture and synthesis of the target organism.

[0227] 2. Strain induction and product determination

[0228] (1) Autotrophic culture of the strain

[0229] Under autotrophic culture conditions, the seed culture was inoculated into 150 mL of culture medium, which was then loaded into 500 mL culture flasks equipped with gas inlets and outlets. A closed-loop gas circulation system driven by a peristaltic pump was used to continuously introduce a mixed gas consisting of 10% CO2, 75% H2, and 15% O2 at a flow rate of 120 mL / h. The gas bag containing the mixed gas was replaced every 6 hours to ensure an adequate gas supply. The culture flasks were placed in a 30°C constant temperature water bath and continuously magnetically stirred for cultivation. After the strain had grown using CO2 for 12 hours, L-arabinose was added to induce TAL expression.

[0230] (2) Induction steps

[0231] Prepare a 100 g / L stock solution of arabinose; add the appropriate amount of arabinose stock solution according to different induction concentrations, as follows:

[0232] 0.1 g / L induction concentration: 150 μL stock solution + 150 mL MSM medium;

[0233] 0.5 g / L induction concentration: 750 μL stock solution + 150 mL MSM medium;

[0234] 1.0 g / L induction concentration: 1500 μL stock solution + 150 mL MSM medium;

[0235] 2.0 g / L induction concentration: 2000 μL stock solution + 150 mL MSM medium;

[0236] The optimal induction concentration was determined based on the coumaric acid yield of the strain at different induction concentrations.

[0237] 3. Detection of coumaric acid

[0238] (1) Take 500 μL of fermentation broth, add 500 μL of ethyl acetate, vortex for 10 mins, centrifuge at 12000 rpm for 10 min, collect ethyl acetate, repeat once, and dry the sample overnight.

[0239] (2) Add 100 μL of methanol to the evaporated centrifuge tube and filter it through an organic filter membrane into the chromatographic bottle.

[0240] (3) The chromatographic vial was placed in a liquid chromatography system for detection. In this embodiment of the invention, the gas chromatography detection method for coumaric acid is as follows: A Thermo Scientific™ UltiMate™ 3000 RS high-performance liquid chromatograph was used. A 5 μm reverse-type column (YMC-Triart C18, 4.6 × 250 mm) was used. The column temperature and sample tray temperature were set to 6 °C. Mobile phase A was 0.1% aqueous acetic acid solution, mobile phase B was 100% acetonitrile, the flow rate was 0.8 mL / min, and the detection wavelength was 310 nm. The elution time was 16 min.

[0241] (4) Results of coumarin acid fermentation test, such as Figure 22 , 23, 24, 25.

[0242] Figure 22 Showing wild type C. necator H16 and PHB synthesis pathway-deficient strain H16△ phaC 1 AB 1 A p-coumaric acid-producing strain was constructed using a chassis. Analysis of the strain's autotrophic growth and p-coumaric acid yield revealed that strain H16△, which lacks the pHB synthesis pathway, is the most effective. phaC 1 AB 1 The engineered strain with the chassis exhibited significantly stronger p-coumaric acid production capacity. After 120 hours of cultivation, the strain's p-coumaric acid yield reached 15.4 mg / L, compared to the wild type. C. necator H16 increased the engineered strain in the chassis by 26.0% (12.2 mg / L).

[0243] Figure 23 This demonstrates the ability to induce the PHB synthesis pathway deletion in strain H16△ using different concentrations of arabinose. phaC 1 AB 1The yield of p-coumaric acid by the engineered strain constructed using a chassis was investigated. Results showed that the strain achieved the highest p-coumaric acid yield (18.7 mg / L) at an arabinose concentration of 2.0%. Further increases in the induction concentration led to a decrease in yield. Therefore, the optimal induction concentration for p-coumaric acid production by the strain was confirmed to be 2.0 mg / L.

[0244] Figure 24 The effects of overexpression on genes involved in the coumaric acid synthesis pathway on coumaric acid production in engineered strains were demonstrated. Results showed that strain H16△, which lacks the PHB synthesis pathway, significantly increased coumaric acid production. phaC 1 AB 1 Based on the engineered strains used to construct the chassis (control group), the synthetic pathway was overexpressed. aroC , aroG1 The strain's p-coumaric acid production reached 27.4 mg / L and 33.0 mg / L, respectively, representing increases of 42.7% and 71.2% compared to the control group (19.2 mg / L), while overexpression of other genes had no promoting effect on yield. Furthermore, [the text abruptly ends here]. aroG1 and aroC Simultaneously, during overexpression, the strain's p-coumaric acid production continued to increase, reaching 42.8 mg / L, indicating that overexpression... aroG1 and aroC It can coordinate and promote the production of p-coumaric acid by the strain. Based on this, [further details are needed]. tal After copying, the p-coumaric acid production of the strain was further increased to 49.3 mg / L, which is 156.1% higher than that of the initial control strain. This indicates that the enhancement of the synthetic pathway effectively increased the p-coumaric acid production of the strain.

[0245] Figure 25 This study demonstrates the effect of overexpression of genes related to NADPH synthesis via genome integration on the production of coumaric acid in engineered strains. Results show that, based on pathway-enhancing strains (control), overexpression of endogenous genes… zwf2 and introduction from E. coli The addition of pntAB increased the coumaric acid production of the strain to 52.7 mg / L and 62.2 mg / L, respectively, representing increases of 10.9% and 30.9% compared to the control group (47.5 mg / L). This indicates that increasing NADPH availability within the engineered strain can effectively promote coumaric acid synthesis.

[0246] Ultimately, through the tyrosine ammonia lyase gene talThe metabolic optimization of the engineered strain was achieved through strategies such as optimizing the induction concentration, optimizing the p-coumaric acid synthesis pathway, and optimizing the supply of intracellular cofactors. This resulted in a 303.9% increase in p-coumaric acid production of the final strain compared to the initial strain. The development of this strain provides a new option for the green production of p-coumaric acid.

[0247] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0248] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0249] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A *Rochelya eutrophic* alkalogenic strain for producing p-coumaric acid, characterized in that, The engineered alkali-producing strain of *Alcaligenes rhodotrophus* was derived from *Alcaligenes rhodotrophus*, by knocking out the phaA, phaB1, and phaC1 genes, and then undergoing any of the following modifications: (1) Transplantation of exogenous gene tal; (2) Transform the exogenous gene tal into the starter strain and overexpress one or more of the endogenous genes aroC and aroG1; (3) Transform the exogenous gene tal, overexpress one or more of the endogenous genes of the starting strain aroC and aroG1, and express the endogenous gene zwf2; (4) Transform the exogenous gene tal, overexpress one or more of the endogenous genes aroC and aroG1 of the starting strain, and transform the exogenous gene pntAB; The nucleotide sequence of the exogenous gene tal is shown in SEQ ID NO. 1; The nucleotide sequence of the exogenous gene pntAB is shown in SEQ ID NO. 5; The nucleotide sequence of the endogenous gene zwf2 is shown in SEQ ID NO.3; The nucleotide sequence of the endogenous gene aroC is shown in SEQ ID NO.13; The nucleotide sequence of the endogenous gene aroG1 is shown in SEQ ID NO.

17.

2. The *Roche* eutrophic alkali-producing engineered strain according to claim 1, characterized in that, The starting strain was *Alcaligenes roximatei* H16.

3. The *Roche* eutrophic alkali-producing engineered strain according to claim 1, characterized in that, The exogenous gene tal is expressed via an inducible promoter. The endogenous genes aroC and aroG1 are expressed via constitutive or inducible promoters. The endogenous gene zwf2 and the exogenous gene pntAB are expressed via a constitutive promoter.

4. The application of the *Rochelya euglenoidis* engineered strain according to any one of claims 1 to 3 in the production of p-coumaric acid.

5. A method for producing p-coumaric acid, characterized in that, The alkali-producing engineered strain of Roche as described in any one of claims 1 to 3 was used for sealed fermentation culture.

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