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

Abstract

The present invention discloses a method for improving the yield and production intensity of 1,3-dihydroxyacetone by Gluconobacter oxydans, belonging to the technical field of fermentation engineering. In the present invention, dehydrogenase genes that potentially affect the metabolic flux of 1,3-dihydroxyacetone are knocked out in Gluconobacter oxydans to enhance the efficiency of converting its substrate glycerol into 1,3-dihydroxyacetone, thereby improving the yield and production intensity of 1,3-dihydroxyacetone. Compared with the control strain G. oxydans WSH-003, the yields, conversion rates, and production intensities of 1,3-dihydroxyacetone 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 are all significantly improved.

Description

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

[0002] The present invention relates to a method for improving the yield and production intensity of dihydroxyacetone by 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, has a sweet and cool taste, is easy to absorb moisture and decompose. 1,3 - Dihydroxyacetone can form a thin film on the skin to prevent water evaporation. Moreover, 1,3 - Dihydroxyacetone can react with the stratum corneum cells on the skin surface to form Schiff base, which will change the color of the skin surface to brown, achieving a similar effect to sun exposure. Therefore, it can be used as a sunscreen in cosmetics. According to this effect, it can also reduce skin diseases that may be caused by excessive ultraviolet radiation, such as vitiligo and leukoderma.

[0004] The production methods of 1,3 - dihydroxyacetone mainly include chemical synthesis method and microbial synthesis method. The main characteristics of the chemical synthesis method are that it is difficult to find a suitable balance between production conditions and original costs. Because when the production conditions are relatively simple, the raw material cost requirements are high, and when the raw material cost is low, the production conditions are demanding. Moreover, precious metals are required in the synthesis process. There are many drawbacks in the production of 1,3 - dihydroxyacetone by this method, mainly including low product purity, many by - products, serious environmental pollution and high product separation and purification costs. Therefore, the use of the chemical method to produce 1,3 - dihydroxyacetone is increasingly restricted. In contrast, the microbial synthesis method is mainly adopted in industry because of its mild reaction conditions, strong specificity and high substrate utilization rate. In addition, the microbial fermentation process has less environmental pollution, the product is relatively easy to separate and purify, and the production cost is also low. From the perspective of technical economy and environmental friendliness, the microbial fermentation method can well avoid the drawbacks of the chemical synthesis method and has relatively greater development potential. Moreover, 1,3 - dihydroxyacetone can use Gluconobacter oxydans as a host in the industrial production process, and produce 1,3 - dihydroxyacetone by converting glycerol through the microbial fermentation method. 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 kinds of polyol dehydrogenases in Gluconobacter oxydans, and generally these dehydrogenases are also called glycerol dehydrogenases. These enzymes can oxidize sugar alcohols with Bertrand-Hudson conformation, such as glycerol, sorbitol, gluconic acid, etc. 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 currently the production of 1,3-dihydroxyacetone from glycerol as a substrate is one of the research hotspots at home and abroad. However, at present, the yield of 1,3-dihydroxyacetone produced from glycerol as a substrate is still low and cannot meet the current industrial production. Summary of the Invention

[0006] Aiming at the problem that the current yield of 1,3-dihydroxyacetone is low and cannot meet the industrial production, in order to further improve the ability of Gluconobacter oxydans to produce 1,3-dihydroxyacetone, a series of recombinant bacteria were constructed by knocking out the dehydrogenase genes potentially related to its metabolic flux, and it was found that the yield, conversion rate and production intensity of 1,3-dihydroxyacetone of the recombinant strains were all higher than those of the control. To solve the above problems, the present invention provides a method for enhancing the production intensity and conversion rate of 1,3-dihydroxyacetone fermentation by knocking out the dehydrogenase genes that affect the metabolic flux of 1,3-dihydroxyacetone.

[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 obtained by knocking out the dehydrogenase genes in Gluconobacter oxydans, and the dehydrogenase genes include genes encoding L-iduronic 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 gluconic acid dehydrogenase G2D.

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

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

[0010] The second object of the present invention is to provide a method for increasing the yield of 1,3-dihydroxyacetone. The genetically engineered bacterium is obtained by knocking out the dehydrogenase genes in Gluconobacter oxydans, and the dehydrogenase genes include the genes encoding L-iduronic 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 gluconic acid dehydrogenase G2D.

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

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

[0013] The third object of the present invention is to provide a method for improving the production intensity of 1,3-dihydroxyacetone by Gluconobacter oxydans, which is characterized by knocking out the dehydrogenase gene in Gluconobacter oxydans; the dehydrogenase gene includes genes encoding L-iduronic acid-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldehyde-ketone dehydrogenase, isocitrate dehydrogenase, NAD(P)H dehydrogenase, zinc-dependent alcohol dehydrogenase, and / or gluconic acid dehydrogenase.

[0014] In one embodiment, the nucleotide sequence of the gene encoding L-iduronic acid-5-dehydrogenase is as shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding NAD-dependent xylitol dehydrogenase is as shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding alcohol dehydrogenase is as shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding aldehyde-ketone dehydrogenase is as shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding isocitrate dehydrogenase is as shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding NAD(P)H dehydrogenase is as shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding zinc-dependent alcohol dehydrogenase is as shown in SEQ ID NO.7; the nucleotide sequence of the gene encoding gluconic acid dehydrogenase is as shown in 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, which uses the genetically engineered bacterium to transform and produce 1,3-dihydroxyacetone.

[0017] In one embodiment, the seed liquid of the genetically engineered bacterium is added to the reaction system, and the reaction is carried out at 25-35 °C and 200-250 rpm for no less than 60 h.

[0018] In one embodiment, the reaction system contains 100 g·L of glycerol -1 , 15-30 g·L of yeast powder -1 , 5.0 g·L of CaCO3 -1 , 1 g·L of MgSO4·7H2O -1 , 2 g·L of (NH4)2SO4 -1 , 0.131 g·L of K2HPO4·3H2O -1 , 0.9 g·L of KH2PO4 -1 , and the pH is 6.2.

[0019] The present invention also provides the application of the genetically engineered bacterium in the production of 1,3-dihydroxyacetone.

[0020] Advantages 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.

[0022] oxydans WSH-003, the yields (g·L -1 ) of 1,3-dihydroxyacetone 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 are increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, 16.49 respectively; the conversion rates (%) are increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, 16.49 respectively; the production intensities (g·L -1 ·h -1 ) are increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23, 0.23 respectively. Description of the Drawings

[0023] Figure 1 It is a diagram showing the effect of knocking out different dehydrogenases in G. oxydans WSH-003 on the yield of 1,3-dihydroxyacetone.

[0024] Figure 2 It is a diagram showing the effect of the control group of knocking out dehydrogenases in G. oxydans WSH-003 on the yield of 1,3-dihydroxyacetone Detailed Embodiments

[0025] (I) Strain: Gluconobacter oxydans WSH-003.

[0026] (II) Types of culture media

[0027] Sorbitol basal medium (g·L -1 ): Sorbitol 40, yeast powder 20. When preparing a solid medium, 20 g / L agar powder needs to be added.

[0028] Seed medium (g·L -1 ): Sorbitol 60, yeast powder 20.

[0029] Fermentation medium (g·L-1 ):100 glycerol, 15 - 30 yeast powder, 5.0 CaCO3, 1 MgSO4·7H2O, 2 (NH4)2SO4, 0.131 K2HPO4·3H2O, 0.9 KH2PO4. Adjust the pH to 6.2 using sulfuric acid.

[0030] (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 is dilute H2SO4 with a concentration of 5 mmol·L -1 , flow rate is 0.5 mL·min -1 , column temperature is 40 °C, injection volume is 10 μL. Ultraviolet detector at 271 nm: Detect the content of 1,3 - dihydroxyacetone. Centrifuge the fermentation broth at 12,000 rpm for 2 min, collect the supernatant, filter it with a 0.22 - μm filter membrane, and then use the Shimadzu liquid chromatograph system to detect the yield of 1,3 - dihydroxyacetone.

[0031] Example 1: Construction of the dehydrogenase knockout cassette

[0032] Amplify the upstream and downstream 1000 - bp sequences of the target gene to be knocked out using the genome of G.oxydans WSH - 003 as a template. At the same time, use primers to amplify the kana gene using pBBR1MCS - 2 as a template, and amplify the upp gene (gene sequence shown in SEQ ID NO.12) using the G.oxydans WSH - 003 genome as a template. Use fusion PCR technology to connect the above four fragments to construct a gene knockout cassette: left homologous arm (HAL) - kana - upp - right homologous arm (HAR), and connect the knockout cassette to the multiple - cloning enzyme - digestion site of the pMD19 - T vector, transform it into Escherichia coli competent cells JM109, and spread the transformants on an LB plate containing kanamycin (kana) (50 mg / L) for screening. Preserve the strains with correct sequencing. Since the dehydrogenase knockout cassette carries the kana (the nucleotide sequence of kana can be found in Genbank: positions 1895 - 2689 of MH539767.1) - upp gene, transform the correctly sequenced dehydrogenase knockout cassette fragment into G.oxydans WSH - 003 to obtain a strain G.oxydans with a defect in the upp gene that can grow normally in a sorbitol minimal medium containing kanamycin kana and cefoxitin (knockout gene::kana - upp). After the first round of screening with kana antibiotic is completed, conduct a second - round screening on a sorbitol minimal medium containing 5 - fluorouracil (300 mg / L) and cefoxitin (50 mg / L) to obtain the target recombinant bacterium.

[0033] Example 2: Construction of recombinant bacterium G. oxydans WSH-1

[0034] The knockout cassette for knocking out the I5D gene was constructed as I5DL-kana-upp-I5DR according to the method of Example 1. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003 and screened by the same method as in Example 1, and the recombinant bacterium G. oxydans WSH-1 with the I5D gene knocked out was obtained.

[0035] Example 3: Construction of recombinant bacterium G. oxydans WSH-2

[0036] The knockout cassette for knocking out the NAD-dependent XD2 gene was constructed as NAD-dependent XD2L-kana-upp-NAD-dependent XD2R according to the method of Example 1. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003 and screened by the same method as in Example 1, and the recombinant bacterium G. oxydans WSH-2 with the NAD-dependent XD2 gene knocked out was obtained.

[0037] Example 4: Construction of recombinant bacterium G. oxydans WSH-3

[0038] The knockout cassette for knocking out the AD4 gene was constructed as AD4L-kana-upp-AD4R according to the method of Example 1. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into G. oxydans WSH-003 and screened by the same method as in Example 1, and the recombinant bacterium G. oxydans WSH-3 with the AD4 gene knocked out was obtained.

[0039] Example 5: Construction of recombinant bacterium G. oxydans WSH-4

[0040] The knockout cassette for knocking out the ASD gene was constructed as ASDL-kana-upp-ASDR according to the method of Example 1. The correctly sequenced dehydrogenase knockout cassette fragment was transformed into Gluconobacter oxydans G. oxydans WSH-003 and screened by the same method as in Example 1, and the recombinant bacterium G. oxydans WSH-4 with the ASD gene knocked out was obtained.

[0041] Example 6: Construction of recombinant bacterium G. oxydans WSH-5

[0042] Construct the knockout cassette for knocking out the ID gene as IDL-kana-upp-IDR according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into Gluconobacter oxydans G.oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant strain G.oxydans WSH-5 with the ID gene knocked out.

[0043] Example 7: Construction of recombinant strain G.oxydans WSH-6

[0044] Construct the knockout cassette for knocking out the NADH-D2 gene as NADH-D2L-kana-upp-NADH-D2R according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into Gluconobacter oxydans G.oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant strain G.oxydans WSH-6 with the NADH-D2 gene knocked out.

[0045] Example 8: Construction of recombinant strain G.oxydans WSH-7

[0046] Construct the knockout cassette for knocking out the Zinc-dependent AD gene as Zinc-dependent AD L-kana-upp-Zinc-dependent AD R according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into Gluconobacter oxydans G.oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant strain G.oxydans WSH-7 with the Zinc-dependent AD gene knocked out.

[0047] Example 9: Construction of recombinant strain G.oxydans WSH-8

[0048] Construct the knockout cassette for knocking out the G2D gene as G2DL-kana-upp-G2DR according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into Gluconobacter oxydans G.oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant strain G.oxydans WSH-8 with the G2D gene knocked out.

[0049] Example 10: Fermentation production of 1,3-dihydroxyacetone by recombinant strain and control strain

[0050] Pick 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 bacterium G. oxydans WSH-003. First, culture them in a sorbitol-based medium for 2-3 days respectively, pick monoclonal colonies and activate them in a seed medium for 24 h. Then, with an inoculum size of 8% (v / v), inoculate the above-activated seed solutions into a fermentation medium respectively and carry out fermentation culture at 30 °C and 220 rpm. When the fermentation lasts for 72 h and the substrate glycerol is consumed completely, the fermentation ends.

[0051] Detect the content of 1,3-dihydroxyacetone in the fermentation broth. The fermentation results are as Figure 1 shown in Table 1. Compared with the control strain G. oxydans WSH-003, the yields (g·L -1 ) of 1,3-dihydroxyacetone 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 are increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, 16.49 respectively; the conversion rates (%) are increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, 16.49 respectively; the production intensities (g·L -1 ·h -1 ) are increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23, 0.23 respectively.

[0052] Table 1 Fermentation results of G. oxydans WSH-003 with different dehydrogenases knocked out

[0053]

[0054] Comparative Example 1

[0055] According to the method of Example 1, genes NADH-DTII (NADH dehydrogenase type II, nucleotide sequence as shown in SEQ ID NO.9), ADLP (Aldehyde dehydrogenase-like protein, nucleotide sequence as shown in SEQ ID NO.10), and NADH-D(Q) (NADH dehydrogenase (quinone), nucleotide sequence as 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, 1,3-dihydroxyacetone was fermentatively produced according to the method of Example 10, and the content of 1,3-dihydroxyacetone was determined. The results are as Figure 2 shown. The results show that the yields, conversion rates, and production intensities of 1,3-dihydroxyacetone of strains G. oxydans WSH-9, G. oxydans WSH-10, and G. oxydans WSH-11 are not significantly improved compared with the control.

[0056] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A genetically engineered bacterium for producing 1,3-dihydroxyacetone, characterized in that, Knock out the alcohol dehydrogenase gene in Gluconobacter oxydans; the nucleotide sequence encoding the alcohol dehydrogenase gene is shown as SEQ ID NO.

3.

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

3. A method for increasing the yield of 1,3-dihydroxyacetone by Gluconobacter oxydans, characterized in that, The method knocks out the alcohol dehydrogenase in Gluconobacter oxydans; the nucleotide sequence encoding the alcohol dehydrogenase gene is shown as SEQ ID NO.

3.

4. The method according to claim 3, wherein The Gluconobacter oxydans uses G. oxydans WSH-003 as the host.

5. A method for improving the production intensity of 1,3-dihydroxyacetone by Gluconobacter oxydans, characterized in that, The method knocks out the alcohol dehydrogenase gene in Gluconobacter oxydans; the nucleotide sequence encoding the alcohol dehydrogenase gene is shown as SEQ ID NO.

3.

6. The method according to claim 5, characterized in that, The Gluconobacter oxydans uses G. oxydans WSH-003 as the host.

7. A method for producing 1,3-dihydroxyacetone, characterized in that Using the genetically engineered bacterium described in claim 1 or 2 to transform and produce 1,3-dihydroxyacetone.

8. The method according to claim 7, characterized in that, Add the seed liquid of the genetically engineered bacterium into the reaction system, react at 25-35 °C and 200-250 rpm, and the reaction time is not less than 60 h.

9. According to the method described in claim 8, the reaction system contains 100 g·L of glycerol -1 , 15 - 30 g·L of yeast powder -1 , 5.0 g·L of CaCO3 -1 , 1 g·L of MgSO4·7H2O -1 , 2 g·L of (NH4)2SO4 -1 , 0.131 g·L of K2HPO4·3H2O -1 , 0.9 g·L of KH2PO4 -1 , and the pH is 6.

2.

10. Use of the genetically engineered bacterium described in claim 1 or 2 in the production of 1,3-dihydroxyacetone.

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