Method for improving the yield and production intensity of 1,3-dihydroxyacetone of Gluconobacter oxydans

Abstract

The present invention discloses a method for improving 1,3-dihydroxyacetone yield and production intensity of Gluconobacter oxydans, and belongs to the field of fermentation engineering technology. The present invention enhances the efficiency of converting its substrate glycerol into 1,3-dihydroxyacetone by knocking out the dehydrogenase gene that has a potential impact on the metabolic flux of 1,3-dihydroxyacetone in Gluconobacter oxydans, thereby improving the yield and production intensity of 1,3-dihydroxyacetone. Compared with the control strain G.oxydansWSH-003, the 1,3-dihydroxyacetone yield, conversion rate and production intensity of recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 are significantly improved.

Description

[0001] This application is a divisional application with an application date of April 25, 2021 and application number 202110445471.3. Technical Field

[0002] The invention relates to a method for improving the yield and production intensity of 1,3-dihydroxyacetone of Gluconobacter oxydans, and belongs to the technical field of fermentation engineering. Background Art

[0003] 1,3-Dihydroxyacetone (DHA) is the simplest three-carbon ketose. It appears as white or milky white powdery crystals with a sweet and cool taste. It easily absorbs moisture and decomposes. 1,3-Dihydroxyacetone can form a thin film on the skin to prevent water evaporation. 1,3-Dihydroxyacetone also undergoes a Schiff base reaction with the stratum corneum cells on the surface of the skin, causing the surface color of the skin to turn brown. This can achieve an effect similar to sun exposure, and therefore, it can be used as a sunscreen in cosmetics. This effect can also reduce skin diseases that may be caused by excessive ultraviolet radiation, such as white spots and vitiligo.

[0004] The main methods for producing 1,3-dihydroxyacetone include chemical synthesis and microbial synthesis. The main drawback of chemical synthesis is the difficulty in finding an appropriate balance between production conditions and initial costs. This is because relatively simple production conditions require high raw material costs, while low raw material costs require high production conditions. Furthermore, the synthesis process requires the use of precious metals. This method for producing 1,3-dihydroxyacetone has numerous drawbacks, including low product purity, numerous byproducts, severe environmental pollution, and high product separation and purification costs. Consequently, the use of chemical methods for producing 1,3-dihydroxyacetone is becoming increasingly limited. In comparison, microbial synthesis is primarily used in industry due to its mild reaction conditions, high specificity, and high substrate utilization. Furthermore, microbial fermentation processes pose minimal environmental risks, the product is relatively easy to separate and purify, and the production cost is relatively low. From the perspective of technological economy and environmental friendliness, microbial fermentation can effectively avoid the drawbacks of chemical synthesis methods and has relatively greater development potential. Moreover, in the industrial production process of 1,3-dihydroxyacetone, glycerol can be converted into 1,3-dihydroxyacetone through microbial fermentation using Staphylococcus oxidans as the host. This method has a simple operation process, is easy to operate and control, has low cost and a short production cycle. Therefore, this method has become the main means of producing 1,3-dihydroxyacetone at home and abroad.

[0005] There are many types of polyol dehydrogenases in Staphylococcus oxydans, which are generally also called glycerol dehydrogenases. This type of enzyme can oxidize sugar alcohols with a Bertrand-Hudson conformation, such as glycerol, sorbitol, and gluconic acid. Most of these enzymes are ethanol, sorbitol, and glycerol dehydrogenases, and their cofactors are mostly pyrroloquinoline quinone (PQQ). Among them, glycerol is the simplest molecule with this conformation, and the production of 1,3-dihydroxyacetone using glycerol as a substrate is currently a hot topic of research both domestically and internationally. However, the current yield of 1,3-dihydroxyacetone using glycerol as a substrate is still low and cannot adapt to current industrial production. Summary of the Invention

[0006] To address the current low 1,3-dihydroxyacetone production, which is insufficient for industrial production, the present invention aims to further enhance the 1,3-dihydroxyacetone production capacity of Staphylococcus oxydans. By deleting dehydrogenase genes potentially related to its metabolic flux, a series of recombinant strains were constructed. Results showed that the recombinant strains exhibited higher 1,3-dihydroxyacetone yields, conversion rates, and production intensity than controls. To address these issues, the present invention provides a method for enhancing the production intensity and conversion rate of 1,3-dihydroxyacetone fermentation by deleting dehydrogenase genes that affect 1,3-dihydroxyacetone metabolic flux.

[0007] The first object of the present invention is to provide a genetically engineered bacterium for producing 1,3-dihydroxyacetone, wherein the genetically engineered bacterium is a knockout of dehydrogenase genes in Gluconobacter oxydans, wherein the dehydrogenase genes include genes encoding L-idonic acid-5-dehydrogenase I5D, NAD-dependent xylitol dehydrogenase NAD-dependent XD2, alcohol dehydrogenase AD4, aldehyde-ketone dehydrogenase ASD, isocitrate dehydrogenase ID, NAD(P)H dehydrogenase NADH-D2, zinc-dependent alcohol dehydrogenase Zinc-dependent AD and / or gluconate dehydrogenase G2D.

[0008] In one embodiment, the nucleotide sequence of the gene encoding L-idonic acid 5-dehydrogenase is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is shown as SEQ ID NO.2; the nucleotide sequence of the gene encoding alcohol dehydrogenase is shown as SEQ ID NO.3; the nucleotide sequence of the gene encoding aldehyde-ketone dehydrogenase is shown as SEQ ID NO.4; the nucleotide sequence of the gene encoding isocitrate dehydrogenase is shown as SEQ ID NO.5; the nucleotide sequence of the gene encoding NAD(P)H dehydrogenase is shown as SEQ ID NO.6; the nucleotide sequence of the gene encoding zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO.7; and the nucleotide sequence of the gene encoding gluconate dehydrogenase is shown as SEQ ID NO.8.

[0009] In one embodiment, G. oxydans WSH-003 is used as the host.

[0010] A second object of the present invention is to provide a method for increasing the production of 1,3-dihydroxyacetone, wherein the genetically engineered bacteria are dehydrogenase genes in Gluconobacter oxydans, wherein the dehydrogenase genes include genes encoding L-idonic acid-5-dehydrogenase I5D, NAD-dependent xylitol dehydrogenase NAD-dependent XD2, alcohol dehydrogenase AD4, aldehyde-ketone dehydrogenase ASD, isocitrate dehydrogenase ID, NAD(P)H dehydrogenase NADH-D2, zinc-dependent alcohol dehydrogenase Zinc-dependent AD and / or gluconate dehydrogenase G2D.

[0011] In one embodiment, the nucleotide sequence of the gene encoding L-idonic acid 5-dehydrogenase is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is shown as SEQ ID NO.2; the nucleotide sequence of the gene encoding alcohol dehydrogenase is shown as SEQ ID NO.3; the nucleotide sequence of the gene encoding aldehyde-ketone dehydrogenase is shown as SEQ ID NO.4; the nucleotide sequence of the gene encoding isocitrate dehydrogenase is shown as SEQ ID NO.5; the nucleotide sequence of the gene encoding NAD(P)H dehydrogenase is shown as SEQ ID NO.6; the nucleotide sequence of the gene encoding zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO.7; and the nucleotide sequence of the gene encoding gluconate dehydrogenase is shown as SEQ ID NO.8.

[0012] In one embodiment, the Gluconobacter oxydans is G. oxydans WSH-003.

[0013] A third object of the present invention is to provide a method for improving the production intensity of 1,3-dihydroxyacetone by Gluconobacter oxydans, characterized in that the dehydrogenase genes in Gluconobacter oxydans are knocked out; the dehydrogenase genes include genes encoding L-idonic acid-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldehyde-ketone dehydrogenase, isocitrate dehydrogenase, NAD(P)H dehydrogenase, zinc-dependent alcohol dehydrogenase and / or gluconate dehydrogenase.

[0014] In one embodiment, the nucleotide sequence of the gene encoding L-idonic acid 5-dehydrogenase is shown as SEQ ID NO.1; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is shown as SEQ ID NO.2; the nucleotide sequence of the gene encoding alcohol dehydrogenase is shown as SEQ ID NO.3; the nucleotide sequence of the gene encoding aldehyde-ketone dehydrogenase is shown as SEQ ID NO.4; the nucleotide sequence of the gene encoding isocitrate dehydrogenase is shown as SEQ ID NO.5; the nucleotide sequence of the gene encoding NAD(P)H dehydrogenase is shown as SEQ ID NO.6; the nucleotide sequence of the gene encoding zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO.7; and the nucleotide sequence of the gene encoding gluconate dehydrogenase is shown as SEQ ID NO.8.

[0015] In one embodiment, the Gluconobacter oxydans is G. oxydans WSH-003.

[0016] The fourth object of the present invention is to provide a method for producing 1,3-dihydroxyacetone, wherein the method comprises utilizing the genetically engineered bacteria to transform and produce 1,3-dihydroxyacetone.

[0017] In one embodiment, the seed liquid of the genetically engineered bacteria is added to the reaction system, and the reaction is carried out at 25-35° C. and 200-250 rpm for a reaction time of not less than 60 h.

[0018] In one embodiment, the reaction system contains 100 g·L glycerol. -1 , Yeast powder 15-30g·L -1 、CaCO35.0g·L -1 、MgSO4·7H2O 1g·L -1 、(NH4)2SO4 2g·L -1 、K2HPO4·3H2O 0.131g·L -1 、KH2PO40.9g·L -1 , pH is 6.2.

[0019] The present invention also provides the use of the genetically engineered bacteria in producing 1,3-dihydroxyacetone.

[0020] Beneficial effects of the present invention:

[0021] The method of the present invention can improve the yield, conversion rate and production intensity of 1,3-dihydroxyacetone. Compared with the control strain G.oxydans WSH-003, the 1,3-dihydroxyacetone yield (g·L) of the recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 was significantly improved. -1 ) increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, and 16.49, respectively; the conversion rate (%) increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, and 16.49, respectively; the production intensity (g·L -1 ·h -1 ) increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23, and 0.23 respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This figure shows the effect of knocking out different dehydrogenases in G.oxydans WSH-003 on the production of 1,3-dihydroxyacetone.

[0023] Figure 2 This is the effect of the dehydrogenase knockout control group on 1,3-dihydroxyacetone production in G.oxydans WSH-003 DETAILED DESCRIPTION

[0024] (1) Strain: G.oxydans WSH-003.

[0025] (2) Culture medium type

[0026] Sorbitol basal medium (g·L -1 ): sorbitol 40, yeast powder 20, add 20g / L agar powder to prepare solid culture medium.

[0027] Seed culture medium (g·L -1 ): Sorbitol 60, yeast powder 20.

[0028] Fermentation medium (g·L -1): Glycerol 100, yeast powder 15-30, CaCO3 5.0, MgSO4·7H2O 1, (NH4)2SO42, K2HPO4·3H2O 0.131, KH2PO4 0.9, and sulfuric acid was used to adjust the pH to 6.2.

[0029] (III) Determination of 1,3-dihydroxyacetone: High performance liquid chromatography (HPLC). Instrument: Agilent 1260 high performance liquid chromatograph, chromatographic conditions: Aminex HPX-87H (Bio-Rad), mobile phase: dilute H2SO4, concentration: 5 mmol / L -1 , flow rate 0.5 mL min -1 The column temperature was 40°C, and the injection volume was 10 μL. UV detection at 271 nm was used to measure 1,3-dihydroxyacetone content. The fermentation broth was centrifuged at 12,000 rpm for 2 minutes. The supernatant was collected and filtered through a 0.22 μm filter. 1,3-dihydroxyacetone production was then measured using a Shimadzu liquid chromatography system.

[0030] Example 1: Construction of dehydrogenase knockout cassette

[0031] The genome of G.oxydans WSH-003 was used as a template to amplify 1000 bp of sequence upstream and downstream of the target gene to be knocked out. At the same time, primers were used to amplify the kana gene using pBBR1MCS-2 as a template, and the upp gene was amplified using the G.oxydans WSH-003 genome as a template (the gene sequence is shown in SEQ ID NO. 12). The four fragments were connected using fusion PCR technology to construct a gene knockout cassette: left homology arm (HAL)-kana-upp-right homology arm (HAR). The knockout cassette was ligated to the multiple cloning restriction site of the pMD19-T vector and transformed into Escherichia coli competent cells JM109. The transformants were plated on LB plates containing kanamycin (50 mg / L) for screening, and the strains with correct sequencing were preserved. Since the dehydrogenase knockout cassette contains the kana (the nucleotide sequence of kana is detailed in Genbank: MH539767.1, positions 1895-2689)-upp gene, the correctly sequenced dehydrogenase knockout cassette fragment was transformed into G.oxydans WSH-003, obtaining an upp gene-deficient strain G.oxydans (knockout gene::kana-upp) that can grow normally in a sorbitol-based medium containing kanamycin kana and cefoxitin. After the first round of kana antibiotic screening was completed, it was subjected to a second round of screening in a sorbitol-based medium containing 5-fluorouracil (300 mg / L) and cefoxitin (50 mg / L), thereby obtaining the target recombinant bacteria.

[0032] Example 2: Construction of recombinant bacteria G.oxydans WSH-1

[0033] According to the method of Example 1, a knockout cassette for knocking out the I5D gene was constructed as I5DL-kana-upp-I5DR, and the correctly sequenced dehydrogenase knockout cassette fragment was transformed into G.oxydansWSH-003. After screening according to the same method as Example 1, the recombinant bacteria G.oxydansWSH-1 with the I5D gene knocked out were obtained.

[0034] Example 3: Construction of recombinant bacteria G.oxydans WSH-2

[0035] According to the method of Example 1, a knockout cassette for knocking out the NAD-dependent XD2 gene was constructed as NAD-dependentXD2L-kana-upp-NAD-dependentXD2R. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G.oxydansWSH-003, and screening was carried out according to the same method as Example 1 to obtain recombinant bacteria G.oxydansWSH-2 with the NAD-dependent XD2 gene knocked out.

[0036] Example 4: Construction of recombinant bacteria G.oxydans WSH-3

[0037] According to the method of Example 1, a knockout cassette for knocking out the AD4 gene was constructed as AD4L-kana-upp-AD4R, and the correctly sequenced dehydrogenase knockout cassette fragment was transformed into G.oxydansWSH-003. After screening according to the same method as Example 1, the recombinant bacteria G.oxydansWSH-3 with the AD4 gene knocked out were obtained.

[0038] Example 5: Construction of recombinant bacteria G.oxydans WSH-4

[0039] According to the method of Example 1, a knockout cassette for knocking out the ASD gene was constructed as ASDL-kana-upp-ASDR. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003, and screening was carried out according to the same method as Example 1 to obtain the recombinant bacteria G. oxydans WSH-4 with the ASD gene knocked out.

[0040] Example 6: Construction of recombinant bacteria G.oxydansWSH-5

[0041] According to the method of Example 1, a knockout cassette for knocking out the ID gene was constructed as IDL-kana-upp-IDR. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003, and screening was carried out according to the same method as Example 1 to obtain the recombinant bacteria G. oxydans WSH-5 with the ID gene knocked out.

[0042] Example 7: Construction of recombinant bacteria G.oxydans WSH-6

[0043] According to the method of Example 1, a knockout cassette for knocking out the NADH-D2 gene was constructed as NADH-D2L-kana-upp-NADH-D2R. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003, and screening was carried out according to the same method as Example 1 to obtain recombinant bacteria G. oxydans WSH-6 with the NADH-D2 gene knocked out.

[0044] Example 8: Construction of recombinant bacteria G.oxydans WSH-7

[0045] According to the method of Example 1, a knockout cassette for knocking out the Zinc-dependent AD gene was constructed as Zinc-dependent ADL-kana-upp-Zinc-dependent AD R. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003, and screening was carried out according to the same method as Example 1 to obtain recombinant bacteria G. oxydans WSH-7 with the Zinc-dependent AD gene knocked out.

[0046] Example 9: Construction of recombinant bacteria G.oxydans WSH-8

[0047] According to the method of Example 1, a knockout cassette for knocking out the G2D gene was constructed as G2DL-kana-upp-G2DR. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003, and screening was carried out according to the same method as Example 1 to obtain the recombinant bacteria G. oxydans WSH-8 with the G2D gene knocked out.

[0048] Example 10: Fermentation production of 1,3-dihydroxyacetone by recombinant bacteria and control bacteria

[0049] The recombinant bacteria G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 prepared in Examples 2-9 and the control bacteria G.oxydans WSH-003 were first cultured in sorbitol basal medium for 2-3 days, and single clones were picked and activated in seed culture medium for 24 hours. Then, the activated cultured seed liquid was inoculated into the fermentation medium at an inoculum amount of 8% (v / v), and fermentation was carried out at 30°C and 220 rpm. After fermentation for 72 hours, the substrate glycerol was consumed and the fermentation was terminated.

[0050] The content of 1,3-dihydroxyacetone in the fermentation broth was tested, and the fermentation results were as follows: Figure 1 As shown in Table 1 , the 1,3-dihydroxyacetone production (g·L) of the recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7, and G.oxydans WSH-8 was higher than that of the control strain G.oxydans WSH-003. -1 ) increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, and 16.49, respectively; the conversion rate (%) increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, and 16.49, respectively; the production intensity (g·L -1 ·h -1 ) increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23, and 0.23 respectively.

[0051] Table 1 Fermentation results of G.oxydans WSH-003 with different dehydrogenase knockouts

[0052]

[0053] Comparative Example 1

[0054] According to the method of Example 1, the genes NADH-DTII (NADHdehydrogenase type II, the nucleotide sequence is shown in SEQ ID NO.9), ADLP (Aldehydedehydrogenase-like protein, the nucleotide sequence is shown in SEQ ID NO.10), and NADH-D (Q) (NADHdehydrogenase (quinone), the nucleotide sequence is shown in SEQ ID NO.11) on the genome of G.oxydans WSH-003 were knocked out respectively to obtain strains G.oxydans WSH-9, G.oxydans WSH-10, and G.oxydans WSH-11. Then, according to the method of Example 10, 1,3-dihydroxyacetone was fermented and produced, and the 1,3-dihydroxyacetone content was determined. The results are as follows: Figure 2 As shown, the results showed that the yield, conversion rate and production intensity of 1,3-dihydroxyacetone of strains G.oxydans WSH-9, G.oxydans WSH-10 and G.oxydans WSH-11 were not significantly improved compared with the control.

[0055] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered bacterium for producing 1,3-dihydroxyacetone, characterized in that: The gene encoding NAD-dependent xylitol dehydrogenase in Gluconobacter oxydans is knocked out; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is shown in SEQ ID NO.

2.

2. The genetically engineered bacterium according to claim 1, characterized in that G.oxydans WSH-003 was used as the host.

3. A method for increasing the yield and / or production intensity of 1,3-dihydroxyacetone of Gluconobacter oxydans, characterized in that: The method knocks out the gene encoding NAD-dependent xylitol dehydrogenase in Gluconobacter oxydans; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is shown in SEQ ID NO.

2.

4. A method for producing 1,3-dihydroxyacetone, characterized in that: The genetically engineered bacteria according to claim 1 or 2 are used to transform and produce 1,3-dihydroxyacetone.

5. The method according to claim 4, characterized in that The seed liquid of the genetically engineered bacteria is added to the reaction system, and the reaction is carried out at 25-35° C. and 200-250 rpm for a reaction time of not less than 60 hours.

6. Use of the genetically engineered bacteria according to claim 1 or 2 in the production of 1,3-dihydroxyacetone.

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