WO2022068026A1 - 一种高效联产3-羟基丙酸和1,3-丙二醇的多菌混合转化体系及建立方法 - Google Patents

一种高效联产3-羟基丙酸和1,3-丙二醇的多菌混合转化体系及建立方法 Download PDF

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WO2022068026A1
WO2022068026A1 PCT/CN2020/131691 CN2020131691W WO2022068026A1 WO 2022068026 A1 WO2022068026 A1 WO 2022068026A1 CN 2020131691 W CN2020131691 W CN 2020131691W WO 2022068026 A1 WO2022068026 A1 WO 2022068026A1
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coli
bacteria
propanediol
gabd4
glycerol
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齐向辉
张宇飞
员君华
张国艳
袁娇
王洋
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Jiangsu University
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    • C12Y102/01024—Succinate-semialdehyde dehydrogenase (NAD+) (1.2.1.24)

Definitions

  • the invention belongs to the technical field of bioengineering, and in particular relates to a multi-bacteria mixed transformation system for co-producing 3-hydroxypropionic acid and 1,3 propanediol with high efficiency and a method for establishing the same.
  • 3-Hydroxypropionic acid and 1,3-propanediol are two industrially important platform compounds that are widely used as precursors of biodegradable polymers and food additives.
  • 3-Hydroxypropionic acid and 1,3-propanediol are produced by chemical synthesis and biological methods. Most of the chemical methods use non-renewable resources as raw materials. The production process consumes a lot of energy, and the products and by-products are difficult to separate and purify. The production process produces immeasurable environmental pollution.
  • Biosynthesis of 3-hydroxypropionic acid and/or 1,3-propanediol mostly uses glucose and glycerol as substrates, and the production of 3-hydroxypropionic acid and 1,3-propanediol using glycerol as a substrate is simple, the research is sufficient, and the raw materials Inexpensive and can solve the problem of excess glycerol.
  • Lactobacillus reuteri has a strong potential for glycerol metabolism, but in the process of metabolizing glycerol to produce 3-hydroxypropionic acid and 1,3-propanediol, it produces 3-hydroxypropionaldehyde, which is cytotoxic and enzymatically toxic, and 3-hydroxypropanal
  • the rate of aldehyde production is much greater than that of 1,3-propanediol.
  • the rapid accumulation of 3-hydroxypropanal is toxic to cells and enzymes, and the reaction stops.
  • the final yield of 3-hydroxypropionic acid and 1,3-propanediol can be improved only if the inhibition of the intermediate metabolite 3-hydroxypropionaldehyde during the transformation of glycerol by Lactobacillus reuteri is relieved. Therefore, it is urgent to design a multi-bacteria mixed transformation system to increase the yield of 3-hydroxypropionic acid and 1,3-propanediol.
  • the present invention provides a multi-bacteria mixed transformation system for co-producing 3-hydroxypropionic acid and 1,3 propanediol with high efficiency and a method for establishing the same.
  • a multi-bacteria mixed transformation system is used to metabolize glycerol, and the system is optimized to rapidly metabolize the toxic intermediate metabolite 3- Hydroxypropionaldehyde.
  • the accumulation of the toxic intermediate metabolite 3-hydroxypropanal was successfully deactivated, allowing the reaction to proceed continuously, maximizing the conversion of glycerol into the final products 3-hydroxypropionic acid and 1,3-propanediol.
  • the present invention achieves the above technical purpose through the following technical means.
  • the invention first provides a multi-bacteria mixed transformation system for co-producing 3-hydroxypropionic acid and 1,3 propanediol with high efficiency, and the system includes Lactobacillus reuteri and genetically engineered Escherichia coli; in terms of mass fraction, the system Among them, Lactobacillus reuteri 25%-75%, genetically engineered Escherichia coli 25%-75%.
  • the genetically engineered Escherichia coli are succinate semialdehyde dehydrogenase engineering Escherichia coli E.coli BL21/pANY-GabD4 (referred to as E.coli GabD4), 1,3-propanediol oxidoreductase engineering Escherichia coli E.coli BL21/pANY-PduQ (referred to as E.coli PduQ) and engineered Escherichia coli E.coli BL21/pANY-GabD4-PduQ (referred to as E.coli) co-expressing succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase one or more of GabD4-PduQ).
  • E.coli GabD4 succinate semialdehyde dehydrogenase engineering Escherichia coli E.coli BL21/pANY-GabD4
  • E.coli PduQ 1,
  • the total concentration of bacterial cells in the system is 10-30 g/L dry cell weight.
  • the system is 50% Lactobacillus reuteri and 50% engineered Escherichia coli expressing succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase in combination, wherein the total bacterial concentration 20g/L cell dry weight.
  • the invention also provides a method for constructing the above-mentioned multi-bacteria mixed transformation system for co-producing 3-hydroxypropionic acid and 1,3 propanediol with high efficiency, which specifically includes the following steps:
  • Lactobacillus reuteri FXZ014 (Lactobacillus reuteri FXZ014) was grown in MRS medium containing 40mM glycerol for 12h, and then the cells were collected by centrifugation at 8000rpm, 4°C, 5min, and washed with 0.1M potassium phosphate buffer (pH 7.0) for 2 After three times, Lactobacillus reuteri resting cells were obtained for use;
  • the genetically engineered Escherichia coli were cultured at 37°C and 220rpm on a shaker until the OD600 was between 0.4 and 0.6, and isopropyl- ⁇ -D-thiogalactoside (IPTG) with a final concentration of 0.5mM was added at 25°C, The target protein expression was induced overnight at 120 rpm, and then the cells were collected by centrifugation at 8000 rpm, 4 °C, and 5 min, respectively, and washed twice with 0.1 M potassium phosphate buffer (pH 7.0) to obtain genetically engineered Escherichia coli resting cells; for use.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Lactobacillus reuteri FXZ014 refers to the literature ZABED H M, ZHANG Y, GUO Q, et al. Co-biosynthesis of 3-hydroxypropionic acid and 1,3-propanediol by a newly isolated Lactobacillus reuteri strain during whole cell biotransformation of glycerol [J]. Journal of Cleaner Production, 2019, 226 (432-42. The method described in the screening.
  • the resting cells of Lactobacillus reuteri prepared above are mixed with genetically engineered Escherichia coli respectively, that is, a multi-bacteria mixed transformation system.
  • Lactobacillus reuteri is 25%-75%
  • genetically engineered Escherichia coli is 25%-75%.
  • the invention also provides the application of the above-mentioned multi-bacteria mixed transformation system, which is to metabolize glycerol to efficiently co-produce 3-hydroxypropionic acid and 1,3-propanediol.
  • the invention adopts a multi-bacteria mixed transformation strategy, and solves the problem of inhibiting the toxic intermediate metabolite 3-hydroxypropionaldehyde in the transformation process of Lactobacillus reuteri single bacteria.
  • the present invention carries out UTR engineering modification to the genetically engineered Escherichia coli expressing succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase jointly in the optimal mixed transformation system, so that the succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase are modified by UTR engineering.
  • - Efficient and balanced expression of propanediol oxidoreductase which solves the problem of coenzyme NAD + and NADH supply in the process of converting 3-hydroxypropionaldehyde to 3-hydroxypropionate and 1,3-propanediol, enabling genetically engineered E. coli to realize coenzyme NAD + and NADH cycle regeneration.
  • the present invention also develops a two-step method adapted to the above-mentioned optimal transformation system and a fed-batch biotransformation technology, which further solves the intermediate product inhibition and coenzyme NAD + and NADH supply problems existing in the current technology, so that the reaction can be carried out continuously and efficiently, Finally, the total output of 3-hydroxypropionic acid and 1,3-propanediol reached 214.39 g/L, the highest level so far, which has broad application prospects and practical significance.
  • Fig. 1 is the production capacity comparison result of different mixed conversion systems (CS) according to the present invention.
  • Figure 2 shows the results of optimization of the transformation conditions of the double-bacteria mixed transformation system CS-2.
  • Figure 3 shows the comparison results of the two-bacteria mixed transformation system CS-2 with different UTR modifications.
  • Figure 4 shows the production capacity of the optimal double-bacteria mixed transformation system CS-2-V3 under different glycerol loads.
  • Figure 5 shows yields at different glycerol concentrations using one-step and two-step transformation techniques.
  • Figure 6 shows the results of production using two fed-batch bioconversion techniques.
  • Figure 7 is a comparison of the production capacity of the transformation systems of single bacteria, double bacteria and three bacteria.
  • Example 1 Construction of genetically engineered Escherichia coli:
  • E.coli GabD4 Succinate semialdehyde dehydrogenase engineering Escherichia coli E.coli BL21/pANY-GabD4 (referred to as E.coli GabD4):
  • the primers were designed using Oligo7.0 software: PANY-F:5'-atgtatatctcctttaaagt-3', PANY-R:5 '-cctccatgggagctcctg-3', GabD4-F1: 5'-taactttaagaaggatatacatatgtaccaagatctggcactgt-3' and GabD4-R1: 5'-tgcaggagctcccatggagggttacgcttgggtgatgaact-3'.
  • PANY-F and PANY-R primer pairs to amplify the pANY1 vector backbone (excluding 6 ⁇ His tag and ccdB expression cassette), PCR reaction parameters: pre-denaturation, 98°C for 1min; denaturation, 98°C for 10s; annealing, 55°C for 10s ; extension, 72°C for 30s; termination extension, 72°C for 5 min; pANY1 vector backbone was obtained after 32 cycles.
  • E.coli PduQ 1,3-Propanediol oxidoreductase engineering Escherichia coli E.coli BL21/pANY-PduQ (referred to as E.coli PduQ):
  • PduQ-F1 5'-taactttaagaaggagatatacatatggaaaatttagtatgccaac-3' and PduQ-R1: 5'-tgcaggagctcccatggaggttaacgaattattgcttcgtaaat-3'.
  • E.coli GabD4-PduQ Engineering Escherichia coli E.coli BL21/pANY-GabD4-PduQ (referred to as E.coli GabD4-PduQ) expressing succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase jointly:
  • step (1) The other steps are basically the same as in step (1), to obtain the engineered Escherichia coli E.coli BL21/pANY-GabD4-PduQ co-expressing succinate semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase.
  • Lactobacillus reuteri FXZ014 was cultured in MRS medium (10g/L tryptone, 10g/L beef extract, 5g/L yeast powder, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L citric acid) Diammonium hydrogen, 0.1 g/L magnesium sulfate, 0.15 g/L manganese sulfate, 1 g/L Tween 80, and 20 g/L glucose) overnight at 37 °C for activation, followed by 40 mM glycerol in MRS medium at 37 °C Expand the culture under anaerobic conditions for 12 hours; then freeze the centrifuged bacterial solution at 8000 rpm and 4 °C for 5 minutes, collect the cells, and discard the supernatant; finally, wash with 0.1 M potassium phosphate buffer (pH 7.0) and centrifuge to obtain Lactobacillus reuteri FXZ014 Resting cells, spare.
  • E.coli GabD4 Three strains of E.coli GabD4, E.coli PduQ and E.coli GabD4-PduQ were activated in LB medium (10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder) overnight respectively, The volume was then expanded with 1% (v/v) inoculum at 37°C, 220rpm, IPTG was OD600 of 0.4-0.6, resulting in a final IPTG concentration of 0.5mM, followed by overnight induction of protein expression at 25°C, 150rpm Then 8000rpm, 4 °C of freezing centrifugation bacterial liquid 5min, collect cells, discard supernatant; Finally wash with 0.1M potassium phosphate buffer (pH 7.0), centrifuge, respectively obtain E.coli GabD4 resting cells, E.coli PduQ Resting cells and E.coli GabD4-PduQ resting cells, set aside.
  • LB medium 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast
  • the overnight activated strains were inoculated into 5 conical flasks containing 50 mL of medium with 1% inoculum respectively, and cultivated under appropriate conditions; the OD600 of one of the flasks of culture was measured every 3 hours, and after recording , the cells were collected by centrifugation, and the cells were washed twice with 0.1M potassium phosphate buffer (pH 7.0), the supernatant was discarded, and the water was dried in an oven until the cell weight did not change; 5 different OD600 values and corresponding Dry cell weight (CDW), through the linear fitting tool, the approximate relationship between OD600 and CDW was obtained: 1OD600 ⁇ 0.34g/L CDW.
  • CDW Dry cell weight
  • Example 3 Establishment and screening of multi-bacteria mixed transformation system
  • the multi-bacteria mixed transformation system after adding different bacteria was investigated to metabolize glycerol to co-produce 3-hydroxypropionic acid and 1,3-propanediol, so as to screen out the optimal multi-bacteria mixed transformation system.
  • the bacteria of different types and contents were mixed to obtain a multi-bacteria mixed transformation system.
  • the bacteria of different types and contents were added to the test groups 1 to 6 respectively.
  • the specific formula is shown in Table 1.
  • the multi-bacteria mixed transformation systems of different formulations in the experimental groups 1 to 6 were transformed into 30 g/L glycerol solution, respectively, and the samples were taken after 4 hours to determine the 3-HP and 1 , 3-PD concentration, the determination conditions are: Aminex HPX-87H (300 ⁇ 7.8mm) chromatographic column, differential refractive index detector, flow rate 0.6mL/min, column temperature 65 °C.
  • Figure 1 shows the measurement results. It can be seen from Figure 1 that the total production of 3-HP and 1,3-PD in the double-bacteria mixed transformation system (CS-2, CS-4, CS-6) is significantly higher than that under the same conditions.
  • Embodiment 4 Condition optimization of double bacteria mixed transformation system CS-2
  • Example 3 the effects of different cell dry weights (20, 25 and 30 g/L), substrate concentrations (40, 50 and 60 g/L) and transformation times (3, 4 and 5 h) on the mixed transformation system CS-2 were investigated respectively. Influence, the mixed transformation system CS-2 obtained in Example 3 was further optimized.
  • Figure 2 shows the test results.
  • the results show that the production of 3-HP and 1,3-PD increased significantly under all conditions before 4h, and almost no change after 4h; when the dry cell weight was 20g/L and the glycerol concentration was 60g /L, the yield of the mixed transformation system reached the maximum value of 45.15g/L, in which the concentrations of 3-HP and 1,3-PD were 26.33 and 18.82g/L, respectively.
  • Embodiment 5 UTR engineering optimizes double bacteria mixed transformation system
  • UTR engineering bacteria with different GabD4 expression levels were used to replace E.coli GabD4-PduQ to further optimize the double-bacteria mixed transformation system CS-2 obtained in Example 3.
  • the 5 kinds of UTR engineering bacteria were respectively E.coli V1, E.coli V2, E.coli V3, E.coli V4, and E.coli V5 were obtained through UTR engineering and genetic engineering, and their GabD4 expression levels ranged from weak to strong.
  • Example 2 L.reuteri FXZ014 resting cells and E.coli V1, E.coli V2, E.coli V3, E.coli V4 and E.coli V5 resting cells were respectively 1:1 The ratio of , and then obtain the following systems CS-2-V1, CS-2-V2, CS-2-V3, CS-2-V4 and CS-2-V5.
  • the experiment was carried out according to the optimal conditions in Example 4: dry cell weight was 20 g/L, glycerol concentration was 60 g/L, pH was 8.5, and transformation time was 4 h.
  • step (1) the performance of the optimal dual-bacteria system CS-2-V3 obtained in step (1) at higher glycerol loading was investigated, and the one-step and two-step transformation of glycerol co-production 3 was compared at high glycerol loading. -Hydroxypropionic acid and 1,3-propanediol capacity.
  • One-step method The two-bacteria mixed system CS-2-V3 was applied to high substrate glycerol concentration (120, 140 and 160g/L), the dry cell weight was 20g/L, pH 8.5, 30°C, 180rpm after transformation for 4h. concentration of each component.
  • Two-step method collect the one-step transformation reaction solution, adjust the pH to 8.5, add 20g/L fresh resting cells (50% L.reuteri FXZ014+50% E.coli V3) again, and measure the concentration of each component after reacting again for 4 hours .
  • the test results are shown in Figure 5.
  • the one-step transformation of the double-bacteria mixed system CS-2-V3 cannot fully utilize 120g/L and higher concentrations of glycerol, and as the substrate concentration increases, the substrate inhibitory effect and intermediate metabolites The greater the inhibitory effect; the two-step transformation on the basis of the one-step method can fully utilize 120 g/L of glycerol, and finally produce 69.36 and 47.58 g/L of 3-HP and 1,3-PD, and no intermediate product 3 was detected.
  • -HPA When using the two-step method to convert 140g/L glycerol, although the yield was improved compared with 120g/L glycerol, 12.56g/L glycerol still failed to be converted.
  • a higher concentration of glycerol 160 g/L was used, the yield was even lower than that with 140 g/L substrate due to substrate and intermediate product inhibition.
  • the initial conditions were cell dry weight 20 g/L, glycerol 20 g/L, pH 8.5, 30 °C, 180 rpm. After 2 h of transformation, samples were collected to analyze their components, supplemented with 20 g/L of glycerol, and then adjusted to pH 8.5; the above steps were repeated every 2 hours until the final yield no longer changed. The test results are shown in Figure 6a. The strategy of adding low-concentration substrates in batches was adopted. The reaction continued until the 18h was stopped, and a total of 3-HP 94.56g/L and 1,3-PD 64.13g/L were generated.
  • the initial conditions were cell dry weight 10 g/L, glycerol 20 g/L, pH 8.5, 30 °C, 180 rpm. After 2 hours of transformation, samples were collected to analyze their components, supplemented with 20 g/L glycerol and resting cells with a dry cell weight of 3 g/L, and then adjusted the pH to 8.5; the above steps were repeated every 2 hours until the final yield did not change. until.
  • Comparative Example 2 Yield comparison of one-step, two-step, and continuous fed-batch biotransformation

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Abstract

提供了一种高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系及建立方法，属于生物工程技术领域；其中，采用多菌混合转化体系来代谢甘油，并对该体系进行优化，通过引入高效代谢3-羟基丙醛的基因工程菌，快速代谢罗伊氏乳杆菌生成的毒性中间代谢产物3-羟基丙醛，从而最大限度地将甘油转化成最终产物3-羟基丙酸和1,3-丙二醇。

Description

一种高效联产3-羟基丙酸和1,3-丙二醇的多菌混合转化体系及建立方法 技术领域
本发明属于生物工程技术领域，具体涉及一种高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系及建立方法。
背景技术
3-羟基丙酸和1,3-丙二醇是工业上两种重要的平台化合物，作为生物可降解性聚合物的前体物质和食品添加剂广泛使用。3-羟基丙酸和1,3-丙二醇的生产有化学合成法和生物法两种。化学法多以不可再生资源作为原料，其生产过程能耗大，产物副产物多难以分离纯化，生产过程产生不可估量的环境污染。生物法合成3-羟基丙酸和/或1,3-丙二醇多以葡萄糖和甘油作为底物，其中以甘油作为底物生产3-羟基丙酸和1,3-丙二醇步骤简单，研究充分，原料廉价，且能解决甘油过剩的问题。
目前，由于生物合成3-羟基丙酸的过程需要消耗辅酶NAD +产生NADH，1,3-丙二醇的生产恰恰相反，将NADH转变为NAD +，只能将3-羟基丙酸和1,3-丙二醇的生产分开进行，否则会造成微生物体内辅酶的不平衡，影响反应持续进行，导致最终产量低。
罗伊氏乳杆菌具有强大的甘油代谢潜力，但其代谢甘油生产3-羟基丙酸和1,3-丙二醇的过程中会产生具有细胞和酶毒性的3-羟基丙醛，且3-羟基丙醛的产生速率远大于1,3-丙二醇。3-羟基丙醛的快速积累对细胞和酶系造成毒害，反应也随之停止。只有解除罗伊氏乳杆菌转化甘油过程中间代谢物3-羟基丙醛抑制问题，才能提升3-羟基丙酸和1,3-丙二醇最终产量。因此，亟需设计出一种多菌混合转化体系来提升3-羟基丙酸和1,3-丙二醇的产量。
发明内容
针对现有技术中存在不足，本发明提供了一种高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系及建立方法。本发明中，采用多菌混合转化体系来代谢甘油，并对该体系进行优化，通过引入高效代谢3-羟基丙醛的基因工程菌，快速代谢罗伊氏乳杆菌生成的毒性中间代谢产物3-羟基丙醛。运用此策略成功解除毒性中间代谢产物3-羟基丙醛的积累，使得反应持续进行，最大限度地将甘油转化成最终产物3-羟基丙酸和1,3-丙二醇。
本发明是通过以下技术手段实现上述技术目的的。
本发明首先提供了一种高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系，该体系中包括罗伊氏乳杆菌和基因工程大肠杆菌；以质量分数计，所述体系中，罗伊氏乳杆菌25％-75％，基因工程大肠杆菌25％-75％。
进一步的，所述基因工程大肠杆菌为琥珀酸半醛脱氢酶工程大肠杆菌E.coli BL21/pANY-GabD4(简称E.coli GabD4)、1,3-丙二醇氧化还原酶工程大肠杆菌E.coli BL21/pANY-PduQ(简称E.coli PduQ)以及联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的工程大肠杆菌E.coli BL21/pANY-GabD4-PduQ(简称E.coli GabD4-PduQ)中的一种或多种。
进一步的，所述体系中菌体总浓度为10-30g/L细胞干重。
进一步的，以质量分数计，所述体系为50％罗伊氏乳杆菌和50％联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的工程大肠杆菌，其中菌体总浓度为20g/L细胞干重。
本发明还提供了上述高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系的构建方法，具体包括如下步骤：
(1)静息细胞的制备：
将罗伊氏乳杆菌FXZ014(Lactobacillus reuteri FXZ014)在含40mM甘油的MRS培养基中生长12h，然后在8000rpm、4℃、5min离心收集菌体，经0.1M磷酸钾缓冲液(pH 7.0)洗涤2次后，得到罗伊氏乳杆菌静息细胞，备用；
将基因工程大肠杆菌分别在37℃、220rpm摇床培养至OD600为0.4-0.6之间时加入终浓度为0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)，25℃、120rpm过夜诱导目标蛋白表达，然后经8000rpm、4℃、5min离心分别收集菌体，经0.1M磷酸钾缓冲液(pH 7.0)洗涤2次后，得到基因工程大肠杆菌静息细胞；备用。
其中，罗伊氏乳杆菌FXZ014参照文献ZABED H M,ZHANG Y,GUO Q,et al.Co-biosynthesis of 3-hydroxypropionic acid and 1,3-propanediol by a newly isolated Lactobacillus reuteri strain during whole cell biotransformation of glycerol[J].Journal of Cleaner Production,2019,226(432-42.中所述方法进行筛选。
(2)高效联产3-羟基丙酸和1,3丙二醇的多菌混合转化体系的建立：
将上述制备的罗伊氏乳杆菌静息细胞分别与基因工程大肠杆菌混合，即得多菌混合转化体系。
在体系中，以质量分数计，罗伊氏乳杆菌25％-75％，基因工程大肠杆菌25％-75％。
本发明还提供了上述多菌混合转化体系的应用，所述应用为代谢甘油高效联产3-羟基丙酸和1,3-丙二醇。
与现有技术相比，本发明的有益效果在于：
本发明采用多菌混合转化策略，解决了罗伊氏乳杆菌单菌转化过程中毒性中间代谢物3-羟基丙醛抑制问题。通过整合能高效转化3-羟基丙醛为3-羟基丙酸或\和1,3-丙二醇的基因工 程菌，建立6个双菌和三菌混合转化体系，缓解了中间代谢物抑制问题。
本发明对最优混合转化体系中联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的基因工程大肠杆菌进行UTR工程修饰，使得其中琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶高效平衡表达，解决了3-羟基丙醛转化为3-羟基丙酸和1,3-丙二醇过程中的辅酶NAD +和NADH供应问题，使得基因工程大肠杆菌自身能够实现辅酶NAD +和NADH的循环再生。
本发明还开发出适配上述最优转化体系的两步法以及分批补料生物转化技术，进一步解决了目前技术存在的中间产物抑制和辅酶NAD +和NADH供应问题，使得反应持续高效进行，最终3-羟基丙酸和1,3-丙二醇的总产量达到214.39g/L，为迄今为止最高水平，具有广阔的应用前景与实际意义。
附图说明
图1为本发明所述不同混合转化体系(CS)生产能力对比结果。
图2为双菌混合转化体系CS-2的转化条件优化结果。
图3为不同UTR修饰的双菌混合转化体系CS-2的比较结果。
图4为最优双菌混合转化体系CS-2-V3在不同甘油负载下的生产能力。
图5为使用一步法以及两步法转化技术在不同甘油浓度下的产量。
图6为采用两种分批补料生物转化技术进行生产的结果。
图7为单菌、双菌以及三菌转化体系的生产能力对比。
具体实施方式
下面结合附图以及具体实施例对本发明作进一步的说明，但本发明的保护范围并不限于此。
实施例1：基因工程大肠杆菌的构建：
(1)琥珀酸半醛脱氢酶工程大肠杆菌E.coli BL21/pANY-GabD4(简称E.coli GabD4)：
根据钩虫贪铜菌编码琥珀酸半醛脱氢酶的基因GabD4序列和载体pANY1上基因元件特征，利用Oligo7.0软件设计引物：PANY-F:5’-atgtatatctccttcttaaagt-3’、PANY-R:5’-cctccatgggagctcctg-3’、GabD4-F1：5’-taactttaagaaggagatatacatatgtaccaagatctggcactgt-3’和GabD4-R1：5’-tgcaggagctcccatggaggttacgcttgggtgatgaact-3’。
使用PANY-F和PANY-R引物对扩增pANY1载体骨架(不包含6×His tag和ccdB表达盒)，PCR反应参数：预变性，98℃1min；变性，98℃10s；退火，55℃10s；延伸，72℃30s；终止延伸，72℃5min；32个循环后得到pANY1载体骨架。
使用GabD4-F1和GabD4-R1引物对扩增含有同源臂的GabD4基因片段，PCR反应参数：预变性，98℃1min；变性，98℃10s；退火，55℃10s；延伸，72℃30s；终止延伸， 72℃5min；32个循环后得到GabD4基因。
将上述两个基因片段按无缝克隆试剂盒说明方法进行重组连接，将pANY1载体骨架与GabD4基因按1:3(mol/mol)混合，加入一倍体积的2×MultiF Seamless Assembly Mix，50℃连接30min；通过热激转化法将重组载体转化E.coli BL21感受态细胞中，将连接产物加入感受态细胞并混匀，冰上放置30min，42℃热激60s，冰上冷却2min后加入900μL LB培养基，37℃孵育1h后涂布含50μg/mL的卡那霉素平板；使用质粒小提试剂盒提取质粒验证其大小是否正确；最终将质粒送苏州金唯智生物公司进行基因测序；结果成功筛选获得重组工程菌株，命名为E.coli BL21/pANY-GabD4，简称E.coli GabD4。
(2)1,3-丙二醇氧化还原酶工程大肠杆菌E.coli BL21/pANY-PduQ(简称E.coli PduQ)：
利用罗伊氏乳杆菌编码1,3-丙二醇氧化还原酶的基因PduQ序列和载体pANY1构建1,3-丙二醇氧化还原酶工程大肠杆菌E.coli BL21/pANY-PduQ，其方法与步骤(1)基本相同，仅有如下区别：
引物：PduQ-F1：5’-taactttaagaaggagatatacatatggaaaaatttagtatgccaac-3’和PduQ-R1：5’-tgcaggagctcccatggaggttaacgaattattgcttcgtaaat-3’。
(3)联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的工程大肠杆菌E.coli BL21/pANY-GabD4-PduQ(简称E.coli GabD4-PduQ)：
利用步骤(1)和(2)中得到的载体pANY-GabD4和pANY-PduQ设计引物：GabD4-F2：5’-cctccatgggagctcctg-3’、GabD4-R2：5’-ttacgcttgggtgatgaactt-3’、PduQ-F2：5’-agttcatcacccaagcgtaatggccttttgctgg-3’和PduQ-R2：5’-tgcaggagctcccatggaggttaacgaattattgcttc-3’。
其他步骤与步骤(1)中基本相同，得到联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的工程大肠杆菌E.coli BL21/pANY-GabD4-PduQ。
实施例2：
(1)静息细胞的制备：
将罗伊氏乳杆菌FXZ014在MRS培养基(10g/L胰蛋白胨、10g/L牛肉浸膏、5g/L酵母粉、2g/L磷酸氢二钾、5g/L乙酸钠、2g/L柠檬酸氢二铵、0.1g/L硫酸镁、0.15g/L硫酸锰、1g/L吐温80和20g/L葡萄糖)中37℃静置过夜活化，接着在含40mM甘油的MRS培养基中37℃厌氧条件下扩大培养12h；然后经8000rpm、4℃冷冻离心菌液5min，收集细胞，弃上清；最后用0.1M磷酸钾缓冲液(pH 7.0)洗涤，离心，得到罗伊氏乳杆菌FXZ014静息细胞，备用。
将E.coli GabD4、E.coli PduQ以及E.coli GabD4-PduQ三种菌株分别经过夜活化于LB培养基(10g/L胰蛋白胨、10g/L氯化钠和5g/L酵母粉)中，然后以1％(v/v)接种量在37℃、 220rpm条件下扩大体积培养，当OD600为0.4-0.6时IPTG，使得IPTG的终浓度为0.5mM，接着25℃、150rpm下过夜诱导蛋白表达；然后经8000rpm、4℃冷冻离心菌液5min，收集细胞，弃上清；最后用0.1M磷酸钾缓冲液(pH 7.0)洗涤，离心，分别得到E.coli GabD4静息细胞、E.coli PduQ静息细胞以及E.coli GabD4-PduQ静息细胞，备用。
(2)静息细胞干重的测量与换算：
将过夜活化的菌种，分别以1％接种量接种于5瓶含50mL对于培养基的锥形瓶中，在适宜条件下进行培养；每隔3小时测量其中一瓶培养物的OD600，记录后，离心收集菌体，并使用0.1M磷酸钾缓冲液(pH 7.0)洗涤细胞两次，弃上清后于烘箱烘干水分，至细胞重量不变为止；最终得到5个不同OD600值以及对应的细胞干重(CDW)，通过线性拟合工具，得到OD600与CDW的近似关系：1OD600≈0.34g/L CDW。
实施例3：多菌混合转化体系的建立及筛选
本实施例中考察了添加了不同菌后的多菌混合转化体系代谢甘油联产3-羟基丙酸和1,3-丙二醇的产量，以此来筛选出最优的多菌混合转化体系。将不同种类和含量的菌混合，获得多菌混合转化体系，试验组1～6中分别添加了不同种类、含量的菌，具体配方如表1所示。
表1.多菌混合转化体系的配方
编号 配方
1 50％罗伊氏乳杆菌FXZ014+25％E.coli GabD4+25％E.coli PduQ
2 50％罗伊氏乳杆菌FXZ014+50％E.coli GabD4-PduQ
3 75％罗伊氏乳杆菌FXZ014+12.5％E.coli GabD4+12.5％E.coli PduQ
4 75％罗伊氏乳杆菌FXZ014+25％E.coli GabD4-PduQ
5 25％罗伊氏乳杆菌FXZ014+37.5％E.coli GabD4+37.5％E.coli PduQ
6 25％罗伊氏乳杆菌FXZ014+75％E.coli GabD4-PduQ
将试验组1～6中不同配方的多菌混合转化体系(细胞干重为15g/L)分别转化30g/L的甘油溶液，4h后分别取样采用高效液相色谱法测定其中3-HP和1,3-PD浓度，其测定条件为：Aminex HPX-87H(300×7.8mm)色谱柱，示差折光检测器，流速0.6mL/min，柱温65℃。图1为测定结果，从图1中可以看出双菌混合转化体系(CS-2、CS-4、CS-6)中3-HP和1,3-PD总产量显著高于相同条件下的三菌混合转化体系(CS-1、CS-3、CS-5)，其中以混合转化系CS-2的3-HP和1,3-PD产量最高，分别达到14.73g/L和10.38g/L。
实施例4：双菌混合转化体系CS-2的条件优化
本实施例中分别考察了不同细胞干重(20、25和30g/L)、底物浓度(40、50和60g/L)以及转化时间(3、4和5h)对混合转化体系CS-2的影响，进一步优化实施例3中所得的混 合转化体系CS-2。
图2为试验结果，结果表明，在4h之前，所有条件下3-HP和1,3-PD产量都明显提升，4h之后几乎不再变化；当细胞干重为20g/L和甘油浓度为60g/L时，混合转化体系产量达到最大值45.15g/L，其中3-HP和1,3-PD浓度分别为26.33和18.82g/L。
实施例5：UTR工程优化双菌混合转化体系
(1)UTR修饰的双菌混合体系建立与筛选：
本实施例中分别采用5种具有不同GabD4表达量的UTR工程菌代替E.coli GabD4-PduQ来进一步优化实施例3中筛选得到的双菌混合转化体系CS-2，5种UTR工程菌分别为E.coli V1、E.coli V2、E.coli V3、E.coli V4和E.coli V5，通过UTR工程和基因工程手段得到，其GabD4表达量由弱到强。
静息细胞制备参照实施例2，将L.reuteri FXZ014静息细胞分别和E.coli V1、E.coli V2、E.coli V3、E.coli V4以及E.coli V5静息细胞按1:1的比例混合，进而得到如下体系CS-2-V1、CS-2-V2、CS-2-V3、CS-2-V4和CS-2-V5。试验按实施例4中的最优条件进行：细胞干重为20g/L，甘油浓度60g/L，pH 8.5，转化时间4h。
试验结果如图3所示，相较双菌体系CS-2，所有经UTR修饰的双菌体系均表现出更高的产量(图3a)，其中由E.coli V3组成的双菌体系CS-2-V3表现最佳，其几乎消耗完60g/L甘油(图3c)，且产生的中间代谢物3-HPA也最少(图3b)，终产物3-HP和1,3-PD产量高达32.37和22.64g/L。
(2)高底物负载下的产量对比：
本步骤中考察了步骤(1)中的得到的最优双菌体系CS-2-V3在更高甘油负载时的表现，比较了在高甘油负载时一步法和两步法转化甘油联产3-羟基丙酸和1，3-丙二醇的能力。一般来说，高的底物浓度是高产的先决条件，且上述最优双菌体系CS-2-V3几乎可以完全消耗60g/L甘油，因此对于双菌体系CS-2-V3进行高浓度底物转化试验，使用更高浓度甘油(70、85、100和120g/L)在pH 8.5，细胞干重20g/L，30℃条件下进行转化试验。
试验结果如图4，更高的甘油负载下3-HP和1,3-丙二醇产量更高(图4a)，但随之也导致更多的甘油残余(图4c)；在高浓度甘油负载下中间代谢物3-HPA积累较多，但差异不显著(图4b)；有趣的是，通过对pH的测定，发现随着反应的进行，pH逐渐降低，单pH降低至6时，反应几乎停止(图4d)。
实施例6：一步法和两步法生物转化
一步法：将双菌混合体系CS-2-V3运用于高的底物甘油浓度(120、140和160g/L),细胞干重20g/L，pH 8.5，30℃，180rpm下转化4h后测定各组分浓度。
两步法：收集一步法转化反应液，调节pH至8.5，再次添加20g/L新鲜静息细胞(50％L.reuteri FXZ014+50％E.coli V3),再次反应4h后测定各组分浓度。
试验结果如图5所示，双菌混合体系CS-2-V3一步法转化不能完全利用120g/L以及更高浓度的甘油，且随着底物浓度增大，底物抑制效应和中间代谢物抑制效应越大；在一步法基础上进行的二步法转化可以完全利用120g/L的甘油，最终产生69.36和47.58g/L的3-HP和1,3-PD，未检测到中间产物3-HPA。使用两步法转化140g/L甘油时，虽然产量较120g/L甘油有所提升，但仍然有12.56g/L甘油未能转化。使用更高浓度的甘油160g/L时，由于底物和中间产物抑制作用，产量甚至低于使用140g/L底物的产量。
实施例7：连续分批补料生物转化
本实施例为了完全解除底物和中间产物抑制作用，针对双菌混合体系CS-2-V3开发了2种分批补料生物转化技术，极大提升了其联产3-HP和1,3-PD的能力。
(1)连续补加甘油
初始条件为细胞干重20g/L，甘油20g/L，pH 8.5,30℃，180rpm。转化2h后，收集样品分析其组分，补加20g/L的甘油，然后调节pH至8.5；每2小时重复以上步骤，直至最终产量不再变化为止。试验结果如图6a所示，采用分批补加低浓度底物的策略，反应持续进行直至18h停止，共生成3-HP 94.56g/L和1,3-PD 64.13g/L。
(2)连续补加甘油和静息细胞
初始条件为细胞干重10g/L，甘油20g/L，pH 8.5,30℃，180rpm。转化2h后，收集样品分析其组分，补加20g/L的甘油以及细胞干重为3g/L的静息细胞，然后调节pH至8.5；每2小时重复以上步骤，直至最终产量不再变化为止。
试验结果如图6b所示，在初始低浓度静息细胞和低浓度底物情况下，几乎解除了中间代谢物和低温抑制现象，新鲜添加的静息细胞也使得反应持续时间更长，最终经24h反应，消耗240g/L甘油，产生3-HP和1,3-PD共计214.39g/L，其中3-HP产量125.93g/L，1,3-PD产量88.46g/L。生产3-HP和1,3-PD强度高达8.93g/L/h，为迄今为止最高水平。
对比例1：单菌、双菌和三菌混合转化甘油产量对比
本实施例中分别考察单菌、双菌、三菌转化甘油产量的差异，分别设定了单一L.reuteri FXZ014 20g/L，双菌L.reuteri FXZ014 10g/L和E.coli BL21/pANY-GabD4-PduQ 10g/L以及三菌L.reuteri FXZ014 10g/L、E.coli BL21/pANY-GabD4 5g/L和E.coli BL21/pANY-PduQ 5g/L转化体系。分别与40g/L底物甘油在30℃、180rpm下反应6h，随后测定3-羟基丙酸、1,3-丙二醇以及甘油残余浓度。
如图7所示，使用单菌进行转化积累了大量中间代谢物3-HPA，仅产生了8.9g/L的3- 羟基丙酸和6.2g/L的1,3-丙二醇，；相对单菌转化，采用多菌混合转化策略，显著缓解了中间产物的抑制作用，提高了3-羟基丙酸和1,3-丙二醇产量。其中，双菌转化的策略效果最佳，产，产生3-羟基丙酸15.3g/L和1,3-丙二醇11.9g/L。
对比例2：一步法、两步法以及连续分批补料生物转化的产量对比
本实施例中分别考察了双菌体系CS-2-V3运用不同转化技术联产3-HP和1,3-PD的差异，一步法、两步法分别按前文实施例中的方式进行，分别增加底物甘油浓度，直至产量不再增加为止，结果如图8所示，普通一步法转化，总产量最低，仅有85.2g/L；而本发明开发的两步法，使得总产量提升了36％；此外，本发明开发的两种分批补料生物转化技术更是具有无与伦比的优势，总产量近乎是普通一步法的两倍多，达到214.39g/L，这也是迄今为止的最优水平。
所述实施例为本发明的优选的实施方式，但本发明并不限于上述实施方式，在不背离本发明的实质内容的情况下，本领域技术人员能够做出的任何显而易见的改进、替换或变型均属于本发明的保护范围。

Claims (9)

  1. 一种多菌混合转化体系，其特征在于，该体系中包括罗伊氏乳杆菌和基因工程大肠杆菌；以质量分数计，所述体系中，罗伊氏乳杆菌25％-75％，基因工程大肠杆菌25％-75％。
  2. 根据权利要求1所述的多菌混合转化体系，其特征在于，所述基因工程大肠杆菌为琥珀酸半醛脱氢酶工程大肠杆菌E.coli GabD4、1,3-丙二醇氧化还原酶工程大肠杆菌E.coli PduQ、联合表达琥珀酸半醛脱氢酶和1,3-丙二醇氧化还原酶的工程大肠杆菌E.coli GabD4-PduQ中的一种或多种。
  3. 根据权利要求1所述的多菌混合转化体系，其特征在于，所述体系中菌体总浓度为10-30g/L细胞干重。
  4. 根据权利要求1所述的多菌混合转化体系，其特征在于，以质量分数计，所述体系包括50％罗伊氏乳杆菌和50％基因工程大肠杆菌。
  5. 根据权利要求1～4所述的多菌混合转化体系，其特征在于，体系中，所述基因工程大肠杆菌为E.coli GabD4-PduQ；体系中，包括50％罗伊氏乳杆菌和50％E.coli GabD4-PduQ，菌体的总浓度为20g/L细胞干重。
  6. 权利要求1所述的多菌混合转化体系的构建方法，其特征在于，包括：
    分别制备罗伊氏乳杆菌FXZ014、基因工程大肠杆菌的静息细胞，按比例混合得到多菌混合转化体系。
  7. 根据权利要求6所述的多菌混合转化体系的构建方法，其特征在于，体系中，以质量分数计，罗伊氏乳杆菌FXZ014占比25％-75％，基因工程大肠杆菌占比25％-75％。
  8. 权利要求1所述的多菌混合转化体系在联产3-羟基丙酸和1,3-丙二醇中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述基因工程菌通过代谢甘油联产3-羟基丙酸和1,3-丙二醇。
PCT/CN2020/131691 2020-09-29 2020-11-26 一种高效联产3-羟基丙酸和1,3-丙二醇的多菌混合转化体系及建立方法 Ceased WO2022068026A1 (zh)

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