CN112226397B

CN112226397B - Multi-bacterium mixed transformation system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol and establishment method

Info

Publication number: CN112226397B
Application number: CN202011046932.1A
Authority: CN
Inventors: 齐向辉; 张宇飞; 员君华; 张国艳; 袁娇; 王洋
Original assignee: Jiangsu University
Current assignee: Zhenjiang Baitai Biotechnology Co.,Ltd.
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-05-31
Anticipated expiration: 2040-09-29
Also published as: WO2022068026A1; CN112226397A

Abstract

The invention provides a multi-bacterium mixed conversion system for efficiently coproducing 3-hydroxypropionic acid and 1,3-propanediol and an establishment method thereof, belonging to the technical field of biological engineering; in the invention, a multi-bacterium mixed conversion system is adopted to metabolize glycerol, the system is optimized, and a toxic intermediate metabolite 3-hydroxypropionaldehyde generated by lactobacillus reuteri is rapidly metabolized by introducing genetic engineering bacteria capable of efficiently metabolizing 3-hydroxypropionaldehyde, so that the glycerol is converted into a final product 3-hydroxypropionic acid and 1,3-propanediol to the maximum extent.

Description

Multi-bacterium mixed transformation system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol and establishment method

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a multi-bacterium mixed transformation system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol and an establishment method thereof.

Background

3-hydroxypropionic acid and 1,3-propanediol are two industrially important platform compounds, widely used as precursor materials of biodegradable polymers and food additives. The production of 3-hydroxypropionic acid and 1,3-propanediol can be carried out by both chemical synthesis and biological methods. The chemical method mostly uses non-renewable resources as raw materials, the production process has high energy consumption, most by-products of the products are difficult to separate and purify, and the production process generates immeasurable environmental pollution. The biological method for synthesizing the 3-hydroxypropionic acid and/or the 1,3-propanediol mostly uses glucose and glycerol as substrates, wherein the steps for producing the 3-hydroxypropionic acid and the 1,3-propanediol by using the glycerol as the substrates are simple, the research is sufficient, the raw materials are cheap, and the problem of excess glycerol can be solved.

At present, NAD is consumed as a result of the coenzyme required for the biosynthesis of 3-hydroxypropionic acid⁺Production of NADH, 1,3-propanediol by conversion of NADH to NAD⁺The production of 3-hydroxypropionic acid and 1,3-propanediol can only be carried out separately, otherwise, the coenzyme imbalance in the microorganism can be caused, the reaction is influenced to be continuously carried out, and the final yield is low.

Lactobacillus reuteri has strong glycerol metabolism potential, but 3-hydroxypropionaldehyde with cell and enzyme toxicity is generated in the process of producing 3-hydroxypropionic acid and 1,3-propanediol by metabolizing glycerol, and the generation rate of the 3-hydroxypropionaldehyde is far greater than that of the 1, 3-propanediol. The rapid accumulation of 3-hydroxypropanal poisons cells and enzyme systems and the reaction is stopped. Only when the problem of inhibition of 3-hydroxypropionaldehyde which is an intermediate metabolite in the process of converting the lactobacillus reuteri into the glycerol is solved, the final yield of the 3-hydroxypropionic acid and the 1,3-propanediol can be improved. Therefore, it is highly desirable to design a multi-bacterial mixed transformation system to increase the yield of 3-hydroxypropionic acid and 1, 3-propanediol.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a multi-bacterium mixed conversion system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol and an establishment method thereof. In the invention, a multi-bacterium mixed transformation system is adopted to metabolize glycerol, the system is optimized, and a toxic intermediate metabolite 3-hydroxypropionaldehyde generated by lactobacillus reuteri is rapidly metabolized by introducing genetic engineering bacteria capable of efficiently metabolizing 3-hydroxypropionaldehyde. The accumulation of a toxic intermediate metabolite, namely 3-hydroxypropionaldehyde, is successfully relieved by applying the strategy, so that the reaction is continuously carried out, and the glycerol is converted into a final product, namely 3-hydroxypropionic acid and 1,3-propanediol to the maximum extent.

The present invention achieves the above-described object by the following means.

The invention firstly provides a multi-bacterium mixed transformation system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol, wherein the system comprises lactobacillus reuteri and genetically engineered escherichia coli; in the system, the mass fraction of the lactobacillus reuteri is 25-75%, and the mass fraction of the genetic engineering escherichia coli is 25-75%.

Furthermore, the genetic engineering escherichia coli is one or more of succinic semialdehyde dehydrogenase engineering escherichia coli E, coli BL21/pANY-GabD4 (E.coli GabD4 for short), 1,3-propanediol oxidoreductase engineering escherichia coli E, coli BL21/pANY-PduQ (E.coli PduQ for short) and engineering escherichia coli E, coli BL21/pANY-GabD4-PduQ (E.coli GabD4-PduQ for short) for jointly expressing succinic semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase.

Furthermore, the total concentration of the thalli in the system is 10-30 g/L of dry cell weight.

Further, the system is 50% of lactobacillus reuteri and 50% of engineering escherichia coli jointly expressing succinic semialdehyde dehydrogenase and 1, 3-propylene glycol oxidoreductase, wherein the total concentration of thalli is 20 g/L of dry cell weight.

The invention also provides a construction method of the multi-bacterium mixed conversion system for efficiently coproducing the 3-hydroxypropionic acid and the 1,3-propanediol, which specifically comprises the following steps:

(1) preparation of resting cells:

lactobacillus reuteri FXZ014 (Lactobacillus reuteri FXZ 014) grows for 12 h in MRS culture medium containing 40 mM glycerol, then the thalli are collected by centrifugation at 8000 rpm and 4 ℃ for 5min, and the thalli are washed for 2 times by 0.1M potassium phosphate buffer solution (pH 7.0) to obtain Lactobacillus reuteri resting cells for later use;

carrying out shake culture on genetically engineered escherichia coli at 37 ℃ and 220 rpm respectively until OD600 is between 0.4 and 0.6, adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.5 mM, inducing target protein expression at 25 ℃ and 120rpm overnight, centrifuging at 8000 rpm and 4 ℃ for 5min respectively, collecting thalli, washing for 2 times by 0.1M potassium phosphate buffer solution (pH 7.0) to obtain genetically engineered escherichia coli resting cells; and (5) standby.

Among them, Lactobacillus reuteri FXZ014 refers to ZABED H M, ZHANG Y, GUO Q, et al, Co-biosynthesis of 3-hydroxyproponic acid and 1,3-propanediol by a new isolated Lactobacillus strain recovery of glycerol [ J ]. Journal of Cleaner Production, 2019, 226 (432-42).

(2) The establishment of a multi-bacterium mixed transformation system for efficiently coproducing 3-hydroxypropionic acid and 1,3-propanediol comprises the following steps:

mixing the prepared Lactobacillus reuteri resting cells with genetically engineered Escherichia coli respectively to obtain a multi-bacterium mixed transformation system.

In the system, the weight percentage of the lactobacillus reuteri is 25-75%, and the weight percentage of the genetic engineering escherichia coli is 25-75%.

The invention also provides an application of the multi-bacterium mixed transformation system, which is used for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol by metabolizing glycerol.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts a multi-bacterium mixed transformation strategy, and solves the problem of inhibiting the toxic intermediate metabolite 3-hydroxypropionaldehyde in the single bacterium transformation process of the lactobacillus reuteri. By integrating genetic engineering bacteria capable of efficiently converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid or \ and 1,3-propanediol, 6 double-bacteria and three-bacteria mixed conversion systems are established, and the problem of intermediate metabolite inhibition is relieved.

The invention carries out UTR engineering modification on genetic engineering escherichia coli jointly expressing succinic semialdehyde dehydrogenase and 1, 3-propylene glycol oxidoreductase in an optimal mixed conversion system, so that the succinic semialdehyde dehydrogenase and the 1, 3-propylene glycol oxidoreductase are efficiently and balancedly expressed, and coenzyme NAD in the process of converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid and 1, 3-propylene glycol is solved⁺And NADH supply problem, so that the genetically engineered Escherichia coli can realize coenzyme NAD⁺And cyclic regeneration of NADH.

The invention also develops the method for adapting the optimal transformation systemA two-step method and a fed-batch bioconversion technology, further solves the problems of intermediate product inhibition and coenzyme NAD existing in the prior art⁺And NADH supply, so that the reaction is continuously and efficiently carried out, and finally the total yield of the 3-hydroxypropionic acid and the 1,3-propanediol reaches 214.39 g/L, which is the highest level so far, and the method has wide application prospect and practical significance.

Drawings

FIG. 1 shows the results of comparing the production capacities of different mixed Conversion Systems (CS) according to the present invention.

FIG. 2 shows the result of optimizing the transformation conditions of the dual-bacteria mixed transformation system CS-2.

FIG. 3 shows the results of comparison of different UTR-modified dual-bacterial mixed transformation system CS-2.

FIG. 4 shows the productivity of the optimal dual-strain mixed transformation system CS-2-V3 under different glycerol loads.

FIG. 5 is a graph of the yield at different glycerol concentrations using one-step and two-step conversion techniques.

FIG. 6 shows the results of production using two fed-batch bioconversion techniques.

FIG. 7 is a comparison of the productivity of single, double and triple strain transformation systems.

FIG. 8 is a comparison of the optimal yields using different bioconversion techniques.

Detailed Description

The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.

Example 1: construction of genetically engineered Escherichia coli:

(1) succinic semialdehyde dehydrogenase engineering escherichia coli E, coli BL21/pANY-GabD4 (E. coli GabD4 for short):

gene encoding succinic semialdehyde dehydrogenase from cuppridinella hookeriGabD4Sequence and genetic element characteristics on vector pANY1, primers were designed using Oligo7.0 software: 5'-atgtatatctccttcttaaagt-3' for PANY-F, 5'-cctccatgggagctcctg-3' for PANY-R, GabD 4-F1: 5'-taactttaagaaggagatatacatatgtaccaagatctggcactgt-3' and GabD 4-R1: 5' -tgcaggagctcccatggaggttacgcttgggtgatgaact-3’。

Amplifying pANY1 vector backbone (not containing 6 XHis tag and ccdB expression cassette) using the PANY-F and PANY-R primer pairs, PCR reaction parameters: pre-denaturation at 98 deg.C for 1 min; denaturation, 10s at 98 ℃; annealing at 55 ℃ for 10 s; extension, 30s at 72 ℃; terminating the extension, 5min at 72 ℃; after 32 cycles, the pANY1 vector backbone was obtained.

Amplification of homology arm containing primers GabD4-F1 and GabD4-R1GabD4Gene fragment, PCR reaction parameters: pre-denaturation at 98 deg.C for 1 min; denaturation, 10s at 98 ℃; annealing at 55 ℃ for 10 s; extension, 30s at 72 ℃; terminating the extension at 72 ℃ for 5 min; the GabD4 gene was obtained after 32 cycles.

The two gene fragments are recombined and connected according to the method of the Seamless cloning kit, the pANY1 vector skeleton and the GabD4 gene are mixed according to the ratio of 1:3 (mol/mol), 2 XMultiF Seamless Assembly Mix with one volume is added, and the two gene fragments are connected for 30 min at 50 ℃; transformation of recombinant vectors by Heat shock transformationE.coliAdding the ligation product into a competent cell of BL21, uniformly mixing, placing on ice for 30 min, thermally shocking at 42 ℃ for 60 s, cooling on ice for 2 min, adding 900 mu L LB culture medium, incubating at 37 ℃ for 1 h, and then coating a kanamycin flat plate with the concentration of 50 mu g/mL; extracting plasmids by using a plasmid miniprep kit to verify whether the sizes of the plasmids are correct; finally sending the plasmid to Suzhou Jinweizhi biology company for gene sequencing; successfully screening the result to obtain a recombinant engineering strain namedE.coliBL21/pANY-GabD4, abbreviated as E. coli GabD 4.

(2) 1,3-propanediol oxidoreductase engineering Escherichia coli E, coli BL21/pANY-PduQ (E, coli PduQ for short):

the 1,3-propanediol oxidoreductase engineering Escherichia coli E, coli BL21/pANY-PduQ is constructed by utilizing the gene PduQ sequence of the Lactobacillus reuteri encoding 1,3-propanediol oxidoreductase and the vector pANY1, the method is basically the same as the step (1), and only the following differences exist:

primer: PduQ-F1: 5'-taactttaagaaggagatatacatatggaaaaatttagtatgccaac-3' and PduQ-R1: 5'-tgcaggagctcccatggaggttaacgaattattgcttcgtaaat-3' are provided.

(3) Engineering escherichia coli E, coli BL21/pANY-GabD4-PduQ (E, coli GabD4-PduQ for short) for combined expression of succinic semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase:

designing primers by using the vectors pANY-GabD4 and pANY-PdiQ obtained in the steps (1) and (2): GabD 4-F2: 5'-cctccatgggagctcctg-3', GabD 4-R2: 5'-ttacgcttgggtgatgaactt-3', PduQ-F2: 5'-agttcatcacccaagcgtaatggccttttgctgg-3' and PduQ-R2: 5'-tgcaggagctcccatggaggttaacgaattattgcttc-3' are provided.

The other steps are basically the same as the step (1), and the engineering escherichia coli E, coli BL21/pANY-GabD4-PduQ for jointly expressing the succinic semialdehyde dehydrogenase and the 1,3-propanediol oxidoreductase is obtained.

Example 2:

(1) preparation of resting cells:

lactobacillus reuteri FXZ014 was activated by standing overnight at 37 ℃ in MRS medium (10 g/L tryptone, 10 g/L beef extract, 5 g/L yeast powder, 2 g/L dipotassium hydrogen phosphate, 5 g/L sodium acetate, 2 g/L diammonium hydrogen citrate, 0.1 g/L magnesium sulfate, 0.15 g/L manganese sulfate, 1 g/L Tween 80 and 20 g/L glucose), followed by extensive culture in MRS medium containing 40 mM glycerol at 37 ℃ for 12 h under anaerobic conditions; then freezing and centrifuging the bacterial liquid for 5min at 8000 rpm and 4 ℃, collecting cells, and discarding the supernatant; finally, the cells were washed with 0.1M potassium phosphate buffer (pH 7.0) and centrifuged to obtain resting cells of Lactobacillus reuteri FXZ014 for further use.

Respectively activating three strains of E.coli GabD4, E.coli PduQ and E.coli GabD4-PduQ in an LB culture medium (10 g/L tryptone, 10 g/L sodium chloride and 5 g/L yeast powder) overnight, then culturing in an amplification volume at 37 ℃ and 220 rpm by using 1% (v/v) inoculation amount, and when the OD600 is 0.4-0.6, carrying out IPTG so that the final concentration of the IPTG is 0.5 mM, and then inducing protein expression overnight at 25 ℃ and 150 rpm; then freezing and centrifuging the bacterial liquid for 5min at 8000 rpm and 4 ℃, collecting cells, and removing supernatant; and finally washing with 0.1M potassium phosphate buffer (pH 7.0), and centrifuging to obtain the E.coli GabD4 resting cells, the E.coli PduQ resting cells and the E.coli GabD4-PduQ resting cells for later use.

(2) Measurement and conversion of resting cell dry weight:

inoculating the overnight activated strains into 5 conical flasks containing 50 mL of the culture medium by using the inoculation amount of 1% respectively, and culturing under appropriate conditions; measuring the OD600 of one flask of the culture every 3 hours, recording, centrifugally collecting thalli, washing cells twice by using 0.1M potassium phosphate buffer (pH 7.0), discarding supernatant, and drying water in an oven until the weight of the cells is unchanged; finally, 5 different OD600 values and corresponding dry cell weight (CDW) were obtained, and by means of a linear fitting tool, the approximate relationship between OD600 and CDW was obtained: 1 OD600 ≈ 0.34 g/L CDW.

Example 3: establishment and screening of multi-bacterium mixed transformation system

In the embodiment, the yield of the glycerol metabolism coproduction of 3-hydroxypropionic acid and 1,3-propanediol of the multi-bacterium mixed transformation system added with different bacteria is considered, so that the optimal multi-bacterium mixed transformation system is screened. The method comprises the steps of mixing different types and contents of bacteria to obtain a multi-bacteria mixed conversion system, adding different types and contents of bacteria into test groups 1-6 respectively, and enabling a specific formula to be shown in table 1.

TABLE 1 formulation of multi-bacterium mixed transformation system

Numbering	Formulation of
		1	50% Lactobacillus reuteri FXZ014+ 25% E.coli GabD 4+ 25% E.coli PduQ
2	50% Lactobacillus reuteri FXZ014+ 50% E. coli GabD4-PduQ
		3	75% Lactobacillus reuteri FXZ014+ 12.5% E. coli GabD 4+ 12.5% E. coli PduQ
4	75% Lactobacillus reuteri FXZ014+ 25% E. coli GabD4-PduQ
		5	25% Lactobacillus reuteri FXZ014+ 37.5% E. coli GabD 4+ 37.5% E. coli PduQ
6	25% Lactobacillus reuteri FXZ014+ 75% E. coli GabD4-PduQ

Converting multi-bacterium mixed conversion systems (the dry cell weight is 15 g/L) with different formulas in test groups 1-6 into glycerol solutions of 30 g/L respectively, sampling after 4 hours, and determining the concentrations of 3-HP and 1,3-PD by adopting a high performance liquid chromatography, wherein the determination conditions are as follows: aminex HPX-87H (300X 7.8 mm) column, differential refractometer, flow rate 0.6 mL/min, column temperature 65 ℃. FIG. 1 shows the measurement results, and it can be seen from FIG. 1 that the total yield of 3-HP and 1,3-PD in the two-bacterium mixed transformation system (CS-2, CS-4, CS-6) is significantly higher than that in the three-bacterium mixed transformation system (CS-1, CS-3, CS-5) under the same conditions, wherein the yield of 3-HP and 1,3-PD in the mixed transformation system CS-2 is the highest, and reaches 14.73 g/L and 10.38 g/L respectively.

Example 4: condition optimization of dual-bacterium mixed transformation system CS-2

In this example, the effect of different dry cell 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 was examined, respectively, to further optimize the mixed transformation system CS-2 obtained in example 3.

FIG. 2 is a graph showing that 3-HP and 1,3-PD yields were significantly increased under all conditions before 4 h and almost unchanged after 4 h; the yield of the mixed transformation system reached a maximum of 45.15 g/L when the dry cell weight was 20 g/L and the glycerol concentration was 60 g/L, with 3-HP and 1,3-PD concentrations of 26.33 and 18.82 g/L, respectively.

Example 5: UTR engineering optimization double-bacterium mixed transformation system

(1) Establishing and screening a UTR modified double-bacterium mixed system:

in this embodiment, 5 UTR engineering bacteria with different expression amounts of GabD4 are respectively used to replace e.coli GabD4-PduQ to further optimize the dual-bacteria mixed transformation system CS-2 obtained by screening in embodiment 3, and the 5 UTR engineering bacteria are respectively e.coli V1, e.coli V2, e.coli V3, e.coli V4, and e.coli V5, and are obtained by means of UTR engineering and genetic engineering, and the expression amount of GabD4 is from weak to strong.

Preparation of resting cells referring to example 2, L.reuteri FXZ014 resting cells were mixed with E.coli V1, E.coli V2, E.coli V3, E.coli V4 and E.coli V5 resting cells at a ratio of 1:1, respectively, to obtain the following systems CS-2-V1, CS-2-V2, CS-2-V3, CS-2-V4 and CS-2-V5. The test was carried out under the optimum conditions in example 4: the dry weight of the cells was 20 g/L, the concentration of glycerol was 60 g/L, pH 8.5, and the conversion time was 4 h.

As shown in FIG. 3, all UTR-modified two-strain systems showed higher yield (FIG. 3 a) compared to two-strain system CS-2, in which the two-strain system CS-2-V3 consisting of E. coli V3 showed the best performance, consumed almost 60 g/L of glycerol (FIG. 3 c), produced the intermediate metabolite 3-HPA the least (FIG. 3 b), and yielded the final products 3-HP and 1,3-PD as high as 32.37 and 22.64 g/L.

(2) Yield comparison at high substrate loading:

in the step, the performance of the optimal dual-bacteria system CS-2-V3 obtained in the step (1) at higher glycerol load is considered, and the capacities of converting glycerol into coproduct of 3-hydroxypropionic acid and 1,3-propanediol by a one-step method and a two-step method at high glycerol load are compared. In general, high substrate concentrations are a prerequisite for high yields and the above described optimized two-strain system CS-2-V3 almost completely consumes 60 g/L of glycerol, so for the two-strain system CS-2-V3 high concentration substrate conversion assays were performed using higher concentrations of glycerol (70, 85, 100 and 120 g/L) at pH 8.5, cell dry weight 20 g/L, 30 ℃.

The results are shown in FIG. 4, with higher glycerol loadings resulting in higher yields of 3-HP and 1,3-propanediol (FIG. 4 a), but with higher glycerol residuals (FIG. 4 c); the intermediary metabolite 3-HPA accumulated more but did not differ significantly at high glycerol loading (fig. 4 b); interestingly, by measuring the pH, it was found that the pH gradually decreased as the reaction proceeded, and the reaction almost stopped when the pH alone decreased to 6 (fig. 4 d).

Example 6: one-step and two-step biotransformations

A one-step method: the dual-bacteria mixed system CS-2-V3 is applied to high substrate glycerol concentrations (120, 140 and 160 g/L), cell dry weight 20 g/L, pH 8.5, 30 ℃, and conversion at 180 rpm for 4 h, and then the concentration of each component is determined.

A two-step method: the one-step transformation reaction solution was collected, the pH was adjusted to 8.5, 20 g/L of fresh resting cells (50% L. reuteri FXZ014+ 50% E. coli V3) were added again, and the concentrations of the respective components were measured after reacting for 4 hours again.

The test result is shown in FIG. 5, the glycerol with the concentration of 120 g/L and higher cannot be completely utilized by one-step conversion of the two-bacterium mixed system CS-2-V3, and the substrate inhibition effect and the intermediate metabolite inhibition effect are larger as the substrate concentration is increased; the two-step conversion, carried out on a one-step basis, allows complete use of 120 g/L of glycerol, ultimately yielding 69.36 and 47.58 g/L of 3-HP and 1,3-PD, with no detectable intermediate 3-HPA. When 140 g/L of glycerol is converted by using the two-step method, the yield is improved compared with 120 g/L of glycerol, but 12.56 g/L of glycerol cannot be converted. At higher concentrations of glycerol 160 g/L, the yield was even lower than with 140 g/L substrate due to substrate and intermediate inhibition.

Example 7: continuous fed-batch bioconversion

In order to completely remove the inhibition effect of the substrate and the intermediate product, a 2-batch feeding biotransformation technology is developed for a two-bacterium mixed system CS-2-V3, and the capacity of co-producing 3-HP and 1,3-PD is greatly improved.

(1) Continuous addition of glycerol

The initial conditions were 20 g/L of dry cell weight, 20 g/L of glycerol, pH 8.5, 30 ℃ and 180 rpm. After 2 h of conversion, collecting a sample, analyzing the components, supplementing 20 g/L of glycerol, and then adjusting the pH to 8.5; the above steps were repeated every 2 hours until the final yield was no longer changed. The results of the experiment are shown in FIG. 6a, and the reaction is continued until 18 h stops by using a strategy of adding low-concentration substrate in batches, and 3-HP 94.56 g/L and 1,3-PD 64.13 g/L are generated together.

(2) Continuous supplementation with glycerol and resting cells

The initial conditions were 10 g/L of dry cell weight, 20 g/L of glycerol, pH 8.5, 30 ℃ and 180 rpm. After 2 h of conversion, samples were collected for analysis of their components, supplemented with 20 g/L of glycerol and resting cells with a dry cell weight of 3 g/L, and then the pH was adjusted to 8.5; the above steps were repeated every 2 hours until the final yield was no longer changed.

As shown in FIG. 6b, in the case of the initial low concentration of resting cells and low concentration of substrate, the intermediate metabolite and low temperature inhibition phenomenon were almost released, and the reaction was continued for a longer period of time by the freshly added resting cells, and finally 214.39 g/L was produced by consuming 240 g/L of glycerol and 1, 3-HP and 1,3-PD in total, wherein the yield of 3-HP was 125.93 g/L and the yield of 1,3-PD was 88.46 g/L, after 24 hours of reaction. The strength of 3-HP and 1,3-PD produced is as high as 8.93 g/L/h, which is the highest level so far.

Comparative example 1: comparison of glycerol yields in single, double and triple bacteria mixed transformation

In this example, differences in the yields of glycerol transformed by single, double and triple bacteria were examined, and single L.reuteri FXZ 01420 g/L, double L.reuteri FXZ 01410 g/L and E.coli BL21/pANY-GabD 4-PdiQ 10 g/L and triple L.reuteri FXZ 01410 g/L, E.coli BL21/pANY-GabD 45 g/L and E.coli BL 21/pANY-PdiQ 5 g/L transformation systems were set. Separately reacted with 40 g/L of substrate glycerol at 30 ℃ for 6 h at 180 rpm, followed by determination of the residual concentrations of 3-hydroxypropionic acid, 1,3-propanediol and glycerol.

As shown in FIG. 7, transformation with a single bacterium accumulated a large amount of the intermediate metabolite, 3-HPA, yielding only 8.9 g/L of 3-hydroxypropionic acid and 6.2 g/L of 1, 3-propanediol; compared with single-bacterium transformation, the multi-bacterium mixed transformation strategy is adopted, the inhibition effect of the intermediate product is obviously relieved, and the yield of the 3-hydroxypropionic acid and the 1,3-propanediol is improved. The strategy of double-bacterium transformation has the best effect, and the yield is 15.3 g/L of 3-hydroxypropionic acid and 11.9 g/L of 1, 3-propanediol.

Comparative example 2: yield comparison for one-step, two-step and continuous fed-batch bioconversion

In this example, differences of co-production of 3-HP and 1,3-PD by using different transformation technologies in a dual-bacteria system CS-2-V3 are considered, and the one-step method and the two-step method are performed in the manner described in the previous example, respectively, and the substrate glycerol concentration is increased until the yield is not increased any more, and as a result, as shown in fig. 8, the total yield is the lowest, and is only 85.2 g/L, due to transformation by using a common one-step method; the two-step method developed by the invention improves the total output by 36%; in addition, the two fed-batch biotransformation technologies developed by the invention have the advantage of incomparable ratio, and the total yield is nearly twice as much as that of the common one-step method, reaching 214.39 g/L, which is also the optimal level so far.

The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A multi-bacterium mixed transformation system is characterized in that the system consists of Lactobacillus reuteri and genetically engineered Escherichia coli; in the system, by mass fraction, 25% -75% of lactobacillus reuteri and 25% -75% of genetic engineering escherichia coli are contained; the genetic engineering escherichia coli is a mixture of succinic semialdehyde dehydrogenase engineering escherichia coli E, coli GabD4 and 1,3-propanediol oxidoreductase engineering escherichia coli E, coli PduQ, or engineering escherichia coli E, coli GabD4-PduQ for jointly expressing succinic semialdehyde dehydrogenase and 1,3-propanediol oxidoreductase.

2. The system of claim 1, wherein the total concentration of the bacterial cells in the system is 10-30 g/L of dry cell weight.

3. The multi-bacterial mixed transformation system of claim 1, wherein the system comprises 50% lactobacillus reuteri and 50% genetically engineered escherichia coli by mass fraction.

4. The multi-bacterium mixed transformation system according to any one of claims 1 to 3, wherein in the system, the genetically engineered Escherichia coli is E.coli GabD 4-PduQ; the system comprises 50% of lactobacillus reuteri and 50% of E, coli GabD4-PduQ, and the total concentration of the thalli is 20 g/L of dry cell weight.

5. The method for constructing a multi-bacterial mixed transformation system according to claim 1, comprising:

respectively preparing resting cells of the lactobacillus reuteri FXZ014 and the genetic engineering escherichia coli, and mixing the resting cells according to a proportion to obtain a multi-bacterium mixed transformation system.

6. The method for constructing a multi-bacterium mixed transformation system according to claim 5, wherein in the system, by mass fraction, Lactobacillus reuteri FXZ014 accounts for 25% -75%, and genetically engineered Escherichia coli accounts for 25% -75%.

7. Use of the multi-bacterial mixed transformation system of claim 1 for the co-production of 3-hydroxypropionic acid and 1, 3-propanediol.

8. The use of claim 7, wherein the genetically engineered bacteria co-produce 3-hydroxypropionic acid and 1,3-propanediol by metabolizing glycerol.