Method for increasing 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. By knocking out dehydrogenase genes in Gluconobacter oxydans that potentially affect the metabolic flux of 1,3-dihydroxyacetone, the efficiency of converting its substrate glycerol into 1,3-dihydroxyacetone is enhanced, 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 application 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 a Schiff base reaction, which will turn the color of the skin surface brown, and this can achieve 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, and precious metals are also required to participate 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, due to its mild reaction conditions, strong specificity and high substrate utilization rate, the microbial synthesis method is mainly adopted in industry. In addition, the microbial fermentation process has little 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 for the production of 1,3 - dihydroxyacetone at home and abroad.

[0005] There are many kinds of polyol dehydrogenases in Gluconobacter oxydans. Generally, these dehydrogenases are also called glycerol dehydrogenases. These enzymes can oxidize sugar alcohols with the 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 using glycerol as a substrate is one of the research hotspots at home and abroad. However, at present, the yield of 1,3-dihydroxyacetone produced using glycerol as a substrate is still low and cannot meet the current industrial production requirements. Summary of the Invention

[0006] Aiming at the problem that the current yield of 1,3-dihydroxyacetone is low and cannot meet the requirements of 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 dehydrogenase genes potentially related to its metabolic flux, and it was found that the yield, conversion rate, and production intensity of 1,3-dihydroxyacetone in 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 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 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 in that the dehydrogenase gene in Gluconobacter oxydans is knocked out; 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 is to use 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, and the reaction time is not 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 increase the yield, conversion rate and production intensity of 1,3-dihydroxyacetone. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] 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 DESCRIPTION OF THE INVENTION

[0024] (I) Strain: Gluconobacter oxydans WSH-003.

[0025] (II) Types of culture media

[0026] 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.

[0027] Seed 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, use sulfuric acid to adjust pH=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 , column temperature 40℃, injection volume 10μL. UV detector 271nm: detect 1,3-dihydroxyacetone content. Centrifuge the fermentation broth at 12,000rpm for 2min, collect the supernatant, filter with 0.22μm filter membrane, and then use Shimadzu liquid chromatography system to detect the production of 1,3-dihydroxyacetone.

[0030] Example 1: Construction of dehydrogenase knockout cassette

[0031] The genome of G.oxydansWSH-003 was used as a template to amplify the 1000 bp sequence upstream and downstream of the target gene to be knocked out. At the same time, the primers were used to amplify the kana gene using pBBR1MCS-2 as a template, and the upp gene was amplified using the genome of G.oxydansWSH-003 as a template (the gene sequence is shown in SEQ ID NO.12). The above four fragments were connected using fusion PCR technology to construct a gene knockout box: left homology arm (HAL)-kana-upp-right homology arm (HAR), and the knockout box was connected to the multiple cloning restriction site of the pMD19-T vector, transformed into Escherichia coli competent cells JM109, and the transformants were coated on LB plates containing kanamycin (50 mg / L) for screening, and the strains with correct sequencing were preserved. Since the dehydrogenase knockout box carries the kana (the nucleotide sequence of kana is detailed in Genbank: MH539767.1, positions 1895-2689)-upp gene, the correctly sequenced dehydrogenase knockout box fragment was transformed into G.oxydansWSH-003 to obtain 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, a second round of screening was performed 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 bacterium G. oxydans WSH-1

[0033] Construct the knockout cassette for knocking out the I5D gene as I5DL-kana-upp-I5DR according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into G. oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant bacterium G. oxydans WSH-1 with the I5D gene knocked out.

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

[0035] Construct the knockout cassette for knocking out the NAD-dependent XD2 gene as NAD-dependent XD2L-kana-upp-NAD-dependent XD2R according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into G. oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant bacterium G. oxydans WSH-2 with the NAD-dependent XD2 gene knocked out.

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

[0037] Construct the knockout cassette for knocking out the AD4 gene as AD4L-kana-upp-AD4R according to the method of Example 1. Transform the dehydrogenase knockout cassette fragment with correct sequencing into G. oxydans WSH-003, and screen according to the same method as in Example 1 to obtain the recombinant bacterium G. oxydans WSH-3 with the AD4 gene knocked out.

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

[0039] Construct the knockout cassette for knocking out the ASD gene as ASDL-kana-upp-ASDR 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 bacterium G. oxydans WSH-4 with the ASD gene knocked out.

[0040] Example 6: Construction of recombinant bacterium G. oxydans WSH-5

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

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

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

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

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

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

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

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

[0049] 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 separately in a sorbitol-based medium for 2-3 days, pick monoclonal colonies and activate them in a seed medium for 24 h. Then, inoculate the above-activated seed solutions into a fermentation medium at an inoculation amount of 8% (v / v), and carry out fermentation culture at 30 °C and 220 rpm. When the fermentation reaches 72 h and the substrate glycerol is consumed completely, the fermentation ends.

[0050] Detect the content of 1,3-dihydroxyacetone in the fermentation broth, and 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, and 16.49 respectively; the conversion rates (%) are increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88, and 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, and 0.23 respectively.

[0051] Table 1 Fermentation results of G. oxydans WSH-003 with different dehydrogenases knocked out

[0052]

[0053] Comparative Example 1

[0054] 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 did not increase significantly compared with the control.

[0055] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person 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. Genetically engineered bacteria for producing 1,3-dihydroxyacetone, characterized in that, Knock out the aldehyde-ketone dehydrogenase gene in Gluconobacter oxydans; the nucleotide sequence encoding the aldehyde-ketone dehydrogenase gene is as shown in SEQ ID NO.

4.

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 aldehyde-ketone dehydrogenase gene in Gluconobacter oxydans; the nucleotide sequence encoding the aldehyde-ketone dehydrogenase gene is as shown in SEQ ID NO.

4.

4. The method according to claim 3, characterized in that 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 aldehyde-ketone dehydrogenase gene in Gluconobacter oxydans; the nucleotide sequence encoding the aldehyde-ketone dehydrogenase gene is as shown in SEQ ID NO.

4.

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, wherein Add the seed liquid of the genetically engineered bacterium into the reaction system and react at 25 - 35 °C and 200 - 250 rpm, and the reaction time is not less than 60 h.

9. The method according to claim 8, wherein 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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