Method for producing D-psicose from escherichia coli through glucose fermentation

Abstract

The invention belongs to the field of escherichia coli metabolic processes, and particularly relates to a method for producing D-psicose from escherichia coli through glucose fermentation. Escherichia coli is used as a chassis host bacterium, a pgi gene, an SUMO-alsE fusion gene and an a6PP gene are subjected to overexpression at the same time, then a pfkA gene, a pfkB gene and a rpiA gene are knocked out in sequence, and a recombinant bacterium capable of producing D-psicose through glucose fermentation is obtained; the recombinant bacteria are used for fermentation, and glucose is converted to generate D-psicose.

Description

Technical Field

[0001] The invention belongs to the field of Escherichia coli metabolic process, and particularly relates to a method for producing D-psicose by using glucose fermentation by Escherichia coli. Background Art

[0002] D-Allulose is a rare monosaccharide with great application potential. It has the sensory characteristics of low calories and high sweetness, and exhibits rich physiological functions, including regulating blood sugar metabolism, anti-oxidative stress, anti-inflammatory response, and delaying atherosclerosis. Based on these characteristics, D-allulose is regarded as a promising functional sugar in the food industry and medical health fields.

[0003] Due to the scarcity of D-psicose in nature, the traditional chemical synthesis process is complex and has a high environmental load, and the large-scale preparation of D-psicose still faces significant technical bottlenecks. Therefore, the development of an efficient, green and economical production route has become the core direction of research. Biocatalysis technology has become a key area of ​​research on D-psicose synthesis in recent years due to its advantages such as environmental friendliness, mild catalytic conditions and high selectivity. At present, the most commonly used approach is to convert D-fructose into D-psicose by D-psicose-3-epimerase. However, the reaction process is thermodynamically reversible, resulting in a low conversion rate, which limits the efficiency and cost competitiveness of industrial production. In response to this problem, this study explored an efficient and economical D-psicose biosynthesis system from the multidimensional perspectives of cell factory construction and process optimization. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for producing D-psicose by fermenting glucose in Escherichia coli. A plasmid vector is constructed to establish a biosynthetic pathway from glucose to D-psicose in Escherichia coli, and then the fermentation medium and fermentation conditions are optimized to increase the yield of D-psicose.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for producing D-psicose by fermenting glucose with Escherichia coli, comprising: using Escherichia coli as a chassis host bacterium, simultaneously overexpressing a pgi gene, a SUMO-alsE fusion gene and an a6PP gene, and then knocking out the pfkA gene, the pfkB gene and the rpiA gene in sequence, to obtain a recombinant bacterium capable of producing D-psicose by fermenting glucose; and fermenting with the recombinant bacterium to convert glucose into D-psicose. Among them, the Escherichia coli is E. coli JM109 (DE3); the NCBI gene number of the pgi gene is 948535; the SUMO-alsE fusion gene is obtained by adding a SUMO tag to the 5' end of the alsE gene, and the NCBI gene number of the alsE gene is 948595; the NCBI sequence number of the a6PP gene is BF9343_0892; the NCBI gene number of the pfkA gene is 948412; the NCBI gene number of the pfkB gene is 946230; and the NCBI gene number of the rpiA gene is 947407.

[0006] Furthermore, the construction method of the above recombinant bacteria is as follows: inserting the pgi gene between the NcoⅠ and BamHI restriction sites of the pETDuet vector to obtain the pETDuet-pgi vector; inserting the SUMO-alsE fusion gene between the NcoⅠ and HindⅢ restriction sites of the pRSFDuet vector, and The a6PP gene was inserted between the restriction sites of SUMO and I to obtain the pRSFDuet-SUMO-alsE-a6PP vector; the pETDuet-pgi vector and the pRSFDuet-SUMO-alsE-a6PP vector were simultaneously transformed into E. coli JM109 (DE3) to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP); based on the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP), the pfkA gene, the pfkB gene and the rpiA gene were knocked out in sequence by homologous recombination technology to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) that can produce D-psicose by glucose fermentation.

[0007] Furthermore, the fermentation method is as follows: a single colony of the recombinant bacteria is cultured in 4 mL of LB liquid medium overnight, the obtained culture solution is inoculated into a shake flask containing fermentation medium at an inoculum rate of 2 vol%, and cultured at 37°C and 220 rpm until OD 600 When the value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and then fermentation was carried out at 37 °C and 220 rpm; The fermentation medium is a basic medium supplemented with kanamycin at a final concentration of 50 μg / mL, ampicillin at a final concentration of 100 μg / mL, and glucose at a final concentration of 10 g / L; the basic medium is selected from any one of LB liquid medium, TB liquid medium, 2×YT liquid medium, TSB liquid medium, SOB liquid medium, and TPL liquid medium; and the liquid volume of the shake flask is 50 mL / 250 mL.

[0008] Furthermore, the fermentation method is as follows: the recombinant bacteria are cultured in LB liquid medium in sequence for seed culture and expansion culture, the obtained expansion culture solution is inoculated into a fermentation tank filled with fermentation medium, and cultured at 37°C and 220rpm until the OD 600 When the value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and then fermentation was carried out at 37 °C and 220 rpm; Furthermore, OD 600 The expanded culture fluid with a value of 1±0.5 was inoculated into a 5L fermenter containing 3L fermentation medium at a rate of 4 vol% and cultured at 37°C and 220 rpm until the OD 600 When the pH value was between 0.6 and 0.8, IPTG was added at a final concentration of 0.2 mM, and then fermentation was carried out at 37°C and 220 rpm. During the fermentation, the pH value of the fermentation medium was controlled to be constant at 6.8, and the dissolved oxygen concentration was controlled to be 30% by constant flow feeding. The formula of the fermentation medium is as follows: 40 g / L glucose, 12 g / L peptone, 24 g / L yeast powder, 16.43 g / L potassium dihydrogen phosphate trihydrate, 2.31 g / L potassium dihydrogen phosphate, and the rest is water; The feeding rate is 10 mL / h, and the feeding liquid is fermentation medium.

[0009] The above method is used in producing D-psicose by fermenting glucose in Escherichia coli.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention realizes the efficient synthesis of D-psicose using cheap glucose, is simple to operate, has low pollution, and effectively solves the problems of low reuse rate and high cost of enzyme catalysis. At the same time, the present invention makes the product relatively single by rationally designing the pathway, greatly improving the production efficiency of D-psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a cell factory diagram of the method for producing D-psicose by using glucose fermentation by Escherichia coli according to the present invention.

[0012] Figure 2 : A is the map of the recombinant vector pETDuet-pgi; B is the map of the recombinant vector pRSFDuet-SUMO-alsE-a6PP.

[0013] Figure 3: A is a diagram of the fermentation products of the recombinant bacteria E. coli JM09 (DE3) -pETDuet-pRSFDute; B is a diagram of the fermentation products of the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA).

[0014] Figure 4 :Fermentation products of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) under TB liquid medium conditions.

[0015] Figure 5 :Fermentation products of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) under 2×YT liquid culture medium conditions.

[0016] Figure 6 :Fermentation products of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) under TSB liquid medium conditions.

[0017] Figure 7 :Fermentation products of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) under SOB liquid medium conditions.

[0018] Figure 8 :Fermentation products of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) under TPL liquid medium conditions.

[0019] Fig. 9 :The effect of fermentation medium pH on the production of D-psicose by glucose fermentation of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA). Taking pH 7.0 as the benchmark, the increase amount is the yield at other pH values ​​minus the yield at pH 7.0; the growth rate is (increase amount / yield)*100%.

[0020] Fig.10 : Fermentation product diagram of recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) using a constant flow rate addition strategy in a fermenter. DETAILED DESCRIPTION

[0021] For the convenience of understanding the present invention, the present invention will be described more comprehensively and meticulously in conjunction with the accompanying drawings and preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments. Unless otherwise defined, all professional terms used hereinafter have the same meaning as those skilled in the art generally understand. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

[0022] In the following text, the NCBI gene number of the phosphoglucose isomerase gene pgi gene is 948535, the NCBI gene number of the D-psicose-6-phosphate epimerase gene alsE gene is 948595, the NCBI sequence number of the D-psicose-6-phosphate phosphatase gene a6PP gene is BF9343_0892, the NCBI gene number of the 6-phosphofructokinase gene pfkA gene is 948412, the NCBI gene number of the 6-phosphofructokinase gene pfkB gene is 946230, and the NCBI gene number of the ribose-5-phosphate isomerase gene rpiA gene is 947407; the SUMO-alsE fusion gene is obtained by adding a SUMO tag to the 5' end of the alsE gene to increase the expression level of the AlsE protein; the SUMO tag (UniProtKB: P55853) is derived from Caenorhabditis elegans.

[0023] Embodiment 1: The formula of LB liquid culture medium is: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, and the rest is water; the pH value is 7.0.

[0024] Insert the pgi gene between the NcoⅠ and BamHI restriction sites of the pETDuet vector to obtain the pETDuet-pgi vector ( Figure 2 A). Insert the SUMO-alsE fusion gene between the NcoⅠ and HindⅢ restriction sites of the pRSFDuet vector, and insert the a6PP gene between the NdeI and Kpn I restriction sites to obtain the pRSFDuet-SUMO-alsE-a6PP vector ( Figure 2B). The pETDuet-pgi vector and the pRSFDuet-SUMO-alsE-a6PP vector were simultaneously transformed into E. coli JM109 (DE3) to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP). Based on the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP), the pfkA gene, the pfkB gene, and the rpiA gene were knocked out in sequence using the λ-red homologous recombination technology to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA).

[0025] The pETDuet empty vector and the pRSFDuet empty vector were simultaneously transformed into E. coli JM109 (DE3) to obtain the recombinant bacteria E. coli JM09 (DE3)-pETDuet-pRSFDute, which was used as a control strain.

[0026] Fermentation verification: The recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) and E. coli JM09 (DE3) -pETDuet-pRSFDute single colonies were inoculated into 4 mL of LB liquid medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, respectively, and cultured overnight at 37 ° C, 220 rpm. The obtained culture solution was inoculated into a 250 mL shake flask containing 50 mL of fermentation medium (LB liquid medium as the base medium, on which kanamycin with a final concentration of 50 μg / mL, ampicillin with a final concentration of 100 μg / mL and glucose with a final concentration of 10 g / L) at an inoculum rate of 2 vol%, and cultured at 37 ° C, 220 rpm until the cell OD 600 When the value reached 0.6-0.8, IPTG was added at a final concentration of 0.2 mM, and then the culture was carried out at 37 °C and 220 rpm for 72 h. During the fermentation period, the fermentation product was sampled every 12 h to measure the OD 600 The fermentation products were analyzed by HPLC using Waters Sugar-Pak TM The chromatographic column and differential refractive index detector (RID) were used to determine the contents of glucose and D-psicose in the fermentation products. The mobile phase was water, the flow rate was set at 0.5 mL / min, the column temperature was 85°C, and the retention time was 20 min.

[0027] The results show that Figure 3), D-psicose was produced in the fermentation product of the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA), and the D-psicose yield reached a maximum of 0.72 g / L at 48 h of fermentation; while D-psicose was not detected in the fermentation product of the control strain E. coli JM09 (DE3) -pETDuet -pRSFDute, thus proving that the pathway of the present invention is feasible.

[0028] Embodiment 2: The formula of LB liquid culture medium is: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, and the rest is water; the pH value is 7.0.

[0029] The formula of TB liquid culture medium is: peptone 12g / L, yeast powder 24g / L, dipotassium hydrogen phosphate trihydrate 16.43g / L, potassium dihydrogen phosphate 2.31g / L, and the rest is water; the pH value is 7.0.

[0030] The formula of 2×YT liquid culture medium is: peptone 16g / L, yeast powder 10g / L, sodium chloride 5g / L, and the rest is water; the pH value is 7.0.

[0031] The formula of TSB liquid culture medium is: 15 g / L peptone, 5 g / L soy peptone, 5 g / L sodium chloride, and the rest is water; the pH value is 7.0.

[0032] The formula of SOB liquid culture medium is: peptone 20g / L, yeast powder 5g / L, sodium chloride 0.6g / L, potassium chloride 1.8g / L, magnesium chloride 0.95g / L, magnesium sulfate 1.2g / L, and the rest is water; the pH value is 7.0.

[0033] The formula of TPL liquid culture medium is: peptone 12g / L, yeast powder g / L, disodium hydrogen phosphate dodecahydrate 15.13g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.5g / L, ammonium chloride 1g / L, dipotassium hydrogen phosphate trihydrate 9.82g / L, ammonium ferric citrate 0.3g / L, citric acid 2.1g / L, magnesium sulfate heptahydrate 0.49g / L, and the rest is water; the pH value is 7.0.

[0034] Fermentation verification: A single colony of the recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) was inoculated into 4 mL of LB liquid medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, and cultured overnight at 37°C and 220 rpm. The obtained culture solution was inoculated into a 250 mL shake flask containing 50 mL of fermentation medium (based on the basal medium, kanamycin with a final concentration of 50 μg / mL, ampicillin with a final concentration of 100 μg / mL, and glucose with a final concentration of 10 g / L) at an inoculum volume of 2 vol%, and cultured at 37°C and 220 rpm until the cell OD 600 When the value reached 0.6-0.8, IPTG was added at a final concentration of 0.2 mM, and then the culture was carried out at 37 °C and 220 rpm for 72 h. During the fermentation period, the fermentation product was sampled every 12 h to measure the OD 600 The fermentation products were analyzed by HPLC using Waters Sugar-Pak TM The chromatographic column and differential refractive index detector (RID) were used to determine the content of glucose and D-psicose in the fermentation product, the mobile phase was water, the flow rate was set to 0.5 mL / min, the column temperature was 85°C, and the retention time was 20 min. The basic fermentation medium was selected from any one of TB liquid medium, 2×YT liquid medium, TSB liquid medium, SOB liquid medium, and TPL liquid medium.

[0035] The results show that Figures 4 to 8 ), when TB liquid medium was used as the basic fermentation medium, the yield of D-psicose reached a maximum of 1.41 g / L at 48 h of fermentation; when 2×YT liquid medium was used as the basic fermentation medium, the yield of D-psicose reached a maximum of 0.96 g / L at 36 h of fermentation; when TSB liquid medium was used as the basic fermentation medium, the yield of D-psicose reached a maximum of 0.099 g / L at 72 h of fermentation; when SOB liquid medium was used as the basic fermentation medium, the yield of D-psicose reached a maximum of less than 0.6 g / L at 24 h of fermentation; when TPL liquid medium was used as the basic fermentation medium, the yield of D-psicose reached a maximum of 0.55 g / L at 60 h of fermentation. Therefore, TB liquid medium is the best choice for basic fermentation medium.

[0036] Embodiment 3: The optimal pH for the growth of Escherichia coli is 6.5-7.5. During the fermentation process, if the pH is too high or too low, it will affect the dissociation of substances. For example, acetic acid often exists in the form of acetate in a neutral environment. In addition, it has a close impact on bacterial growth, metabolism, etc. Inappropriate pH will affect growth and even cause the death of the strain. Based on this, the initial pH values ​​of the TB liquid culture medium in Example 2 were adjusted to 6.6, 6.8, 7.0, 7.2 and 7.4 respectively, as the basic fermentation medium, and then fermentation verification was carried out according to the method of Example 2. After the fermentation was completed, the content of D-psicose in the fermentation product was measured.

[0037] The results show that Fig. 9 ), when TB liquid medium with an initial pH of 6.8 was used as the basic fermentation medium, the yield of D-psicose in the fermentation product of the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) was the highest, which was 1.97 g / L.

[0038] Embodiment 4: The formula of LB liquid culture medium is: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, and the rest is water; the pH value is 7.0.

[0039] The formula of the fermentation medium is: 40 g / L glucose, 12 g / L peptone, 24 g / L yeast powder, 16.43 g / L dipotassium hydrogen phosphate trihydrate, 2.31 g / L potassium dihydrogen phosphate, and the rest is water; pH = 6.8.

[0040] A single colony of the recombinant E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain a seed solution. The seed solution was inoculated into LB liquid medium at an inoculum volume of 4 vol% and cultured at 37°C and 220 rpm until OD 600 The expanded culture solution was inoculated into a 5L fermenter containing a 3L fermenter medium at 4 vol% and cultured at 37°C and 220 rpm until OD 600 When the value is 0.6-0.8, add IPTG with a final concentration of 0.2mM, and then ferment and cultivate. Set the fermentation temperature to 37°C, the stirring speed of the fermenter to 220rpm, and the fermentation time to 80h. During the fermentation, keep the pH value of the fermentation medium in the fermenter at 6.8. At the same time, feed the fermentation medium at a constant flow rate to control the dissolved oxygen concentration at 30%. The feed liquid is the fermentation medium, and the feed rate is 10mL / h. Sample the fermentation product every 8h during the fermentation to measure the OD 600The fermentation products were analyzed by HPLC using Waters Sugar-Pak TM The chromatographic column and differential refractive index detector (RID) were used to determine the contents of glucose and D-psicose in the fermentation products. The mobile phase was water, the flow rate was set at 0.5 mL / min, the column temperature was 85°C, and the retention time was 20 min.

[0041] The results show that Fig.10 ), the yield of D-psicose reached a maximum of 6.84 g / L at 48 h of fermentation.

[0042] The above are all preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for producing D-psicose by fermenting glucose using Escherichia coli, characterized in that: Escherichia coli is used as a chassis host bacterium, the pgi gene, the SUMO-alsE fusion gene and the a6PP gene are overexpressed at the same time, and the pfkA gene, the pfkB gene and the rpiA gene are knocked out in sequence, so as to obtain a recombinant bacterium capable of producing D-psicose by glucose fermentation; the recombinant bacterium is used for fermentation to convert glucose into D-psicose.

2. The method according to claim 1, characterized in that: The Escherichia coli is E. coli JM109 (DE3); the NCBI gene number of the pgi gene is 948535; the SUMO-alsE fusion gene is obtained by adding a SUMO tag to the 5' end of the alsE gene, and the NCBI gene number of the alsE gene is 948595; the NCBI sequence number of the a6PP gene is BF9343_0892; the NCBI gene number of the pfkA gene is 948412; the NCBI gene number of the pfkB gene is 946230; and the NCBI gene number of the rpiA gene is 947407.

3. The method according to claim 1, characterized in that: The construction method of the recombinant bacteria is as follows: inserting the pgi gene between the NcoⅠ and BamHI restriction sites of the pETDuet vector to obtain the pETDuet-pgi vector; The SUMO-alsE fusion gene was inserted between the NcoⅠ and HindⅢ restriction sites of the pRSFDuet vector, and the a6PP gene was inserted between the Nde I and Kpn I restriction sites to obtain the pRSFDuet-SUMO-alsE-a6PP vector; the pETDuet-pgi vector and the pRSFDuet-SUMO-alsE-a6PP vector were simultaneously transformed into E. coli JM109 (DE3) to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP); based on the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP), the pfkA gene, the pfkB gene and the rpiA gene were knocked out in sequence by homologous recombination technology to obtain the recombinant bacteria E. coli (pgi, SUMO-alsE, a6PP, ΔpfkA, ΔpfkB, ΔrpiA) that can produce D-psicose by glucose fermentation.

4. The method according to claim 1, characterized in that: The fermentation method comprises: culturing a single colony of the recombinant bacteria in 4 mL of LB liquid medium overnight, inoculating the obtained culture solution into a shake flask containing fermentation medium at an inoculum volume of 2 vol%, and culturing at 37° C. and 220 rpm until OD 600 When the value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and then fermentation was carried out at 37°C and 220 rpm.

5. The method according to claim 4, characterized in that: The fermentation medium is a basal medium supplemented with 50 μg / mL of kanamycin, 100 μg / mL of ampicillin, and 10 g / L of glucose. The basic culture medium is selected from any one of LB liquid culture medium, TB liquid culture medium, 2×YT liquid culture medium, TSB liquid culture medium, SOB liquid culture medium, and TPL liquid culture medium; and the liquid volume of the shaking flask is 50 mL / 250 mL.

6. The method according to claim 1, characterized in that: The fermentation method comprises: performing seed culture and expansion culture of the recombinant bacteria in LB liquid medium in sequence, inoculating the obtained expanded culture solution into a fermentation tank filled with fermentation medium, and culturing at 37° C. and 220 rpm until OD 600 When the value was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and then fermentation was carried out at 37°C and 220 rpm.

7. The method according to claim 6, characterized in that: OD 600 The expanded culture fluid with a value of 1±0.5 was inoculated into a 5L fermenter containing 3L fermentation medium at a rate of 4 vol% and cultured at 37°C and 220 rpm until the OD 600 When the value was 0.6-0.8, IPTG was added at a final concentration of 0.2 mM, and then fermentation was carried out at 37°C and 220 rpm. During the fermentation, the pH value of the fermentation medium was controlled to be constant at 6.8, and the dissolved oxygen concentration was controlled to be 30% by constant flow feeding.

8. The method according to claim 7, characterized in that: The formula of the fermentation medium is: 40 g / L glucose, 12 g / L peptone, 24 g / L yeast powder, 16.43 g / L dipotassium hydrogen phosphate trihydrate, 2.31 g / L potassium dihydrogen phosphate, and the rest is water.

9. The method according to claim 7, characterized in that: The feeding rate is 10 mL / h, and the feeding liquid is the fermentation medium.

10. Use of the method according to any one of claims 1 to 9 in producing D-psicose.

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