GB2594671A - Multi-strain co-culture system for efficiently co-producing 3-hydroxypropionic acid and 1,3-propanediol, and establishment method thereof - Google Patents

Abstract

A multi-strain co-culture system comprising Lactobacillus reuteri (L. reuteri) in mass fraction 25%-75% and genetically engineered E. coli in mass fraction 25%-75% is used to metabolize glycerol. The system is optimized by introducing genetically engineered E. coli bacteria that can efficiently metabolize 3-hydroxypropionaldehyde (3-HPA) by expressing enzymes such as succinic semialdehyde dehydrogenase (SSADH) (E. coli GabD4), 1,3-propanediol (1,3-PD) oxidoreductase (E. coli PduQ), or both, thereby maximizing the conversion of glycerol into the final products of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PD). The 3-HPA is a toxic intermediate metabolite produced during rapid metabolism of Lactobacillus reuteri (L. reuteri), such as with the strain FXZ014.

Description

MULTI-STRAIN CO-CULTURE SYSTEM FOR EFFICIENTLY CO-PRODUCING 3-HYDROXYPROPIONIC ACID AND 1,3-PROPANEDIOL, AND ESTABLISHMENT METHOD THEREOF

Technical Field

The present invention belongs to the technical field of bioengineering, and specifically relates to a multi-strain co-culture system for efficiently co-producing 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PD), and an establishment method thereof

Background

As two important industrial platform chemicals, 3-HP and 1,3-PD are widely used as precursors for biodegradable polymers and as food additives. The 3-HP and 1,3-PD are commonly produced by chemical synthesis and biological synthesis. The chemical synthesis method mostly uses non-renewable resources as raw materials, requires large energy consumption in production, and produces many by-products difficult to separate and purify. This inevitably leads to immeasurable environmental pollution. The biological synthesis of 3-HP and/or 1,3-PD is achieved mostly with glucose and glycerol as substrates. There is sufficient research on the use of glycerol as a substrate to produce 3-HP and 1,3-PD, which involves simple steps and cheap raw materials and can solve the problem of excess glycerol.

At present, since a biological synthesis process of 3-HP requires the transformation of NADinto NADH and the production of 1,3-PD is just the opposite (which requires the transformation of NADH into NAD-), 3-HP and 1,3-PD can only be produced separately, otherwise, NAD-NADH imbalance will appear in microorganisms, which will affect the continuous progress of a reaction, resulting in a low final yield.

Lactobacillus reuteri reuteri) has a strong glycerol metabolism potential, but during a glycerol metabolism process of L. reuteri to produce 3-HP and 1,3-PD, 3-hydroxypropionaldehyde (3-HPA) with cellular and enzymatic toxicity will be produced, and a production rate of 3-HPA is much higher than that of 1,3-PD. The rapid accumulation of 3-HPA is harmful to the cell and enzyme systems, and as a result, the reaction will stop. Only when the inhibition of the intermediate metabolite of 3-HPA in a glycerol conversion process of L. reuteri is eliminated, the final yields of 3-HP and 1,3-PD can be improved. Therefore, it is urgent to design a multi-strain co-culture system to improve the yields of 3-HP and 1,3-PD.

Summary

In view of the shortcomings in the prior art, the present invention provides a multi-strain co-culture system for efficiently co-producing 3-HP and 1,3-PD, and an establishment method thereof In the present invention, a multi-strain co-culture system is used to metabolize glycerol, and the system is optimized by introducing genetically engineered bacteria that can efficiently metabolize 3-HPA. The 3-HPA is a toxic intermediate metabolite produced during rapid metabolism of L. reuteri. The use of the strategy can successfully eliminate the accumulation of the toxic intermediate metabolite 3-HPA, such that a reaction can continuously proceed and the conversion of glycerol into the final products of 3-HP and 1,3-PD is maximized.

The present invention achieves the above technical objectives through the following technical means.

The present invention provides a multi-strain co-culture system for efficiently co-producing 3-HP and 1,3-PD, including L. reuleri and genetically engineered Esztherichia coli (E. colt). In the system, the L. reuteri has a mass fraction of 25% to 75% and the genetically engineered E. colt has a mass fraction of 25% to 75%.

Further, the genetically engineered E. colt is one or more from the group consisting of E. colt expressing succinic semialdehyde dehydrogenase (SSADH) (E. co/i BL21/pANY-GabD4, short for E. colt GabD4), E. coil expressing 1,3-PD oxidoreductase (E. colt BL21/pANY-PduQ, short for E. colt PduQ), and E. colt co-expressing the SSADH and the 1,3-PD oxidoreductase (E. colt BL21/pANY-GabD4-PduQ, short for E. colt GabD4-PduQ).

Further, the bacteria in the system have a total concentration of 10 g/L to 30 g/L based on cell dry weight (CDW).

Further, the system includes L. reuteri with a mass fraction of 50% and E. colt GabD4-PduQ with a mass fraction of 50%, in which case the bacteria have a total concentration of 20 g/L based on CDW.

The present invention also provides an establishment method of the multi-strain co-culture system for efficiently co-producing 3-HP and 1,3-PD, specifically including the following steps: (1) preparation of resting cells: cultivating the L. reuteri FXZ014 in an MRS medium with 40 Mkt glycerol for 12 h, centrifuging a resulting culture at 8,000 rpm and 4°C for 5 min to collect bacterial cells, and washing the bacterial cells twice with a 0.1 M potassium phosphate buffer (pH 7.0) to obtain resting cells of the L. reuteri for later use; cultivating the genetically engineered E. colt on a shaker at 37°C and 220 rpm until 0D600 is between 0.4 and 0.6, adding isopropyl-f3-D-thiogalacto side (WIG) at a final concentration of 0.5 mM, and incubating a resulting mixture overnight at 25°C and 120 rpm to induce the expression of a target protein; and centrifuging a resulting culture at 8,000 rpm and 4°C for 5 min to collect bacterial cells, and washing the bacterial cells twice with a 0.1 M potassium phosphate buffer (pH 7.0) to obtain resting cells of the genetically engineered E. colt for later use; wherein the L. renter! FXZ014 is screened with reference to the method described in the literature ZABED H NI, ZHANG Y, GUO Q, et al. Co-biosynthesis of 3-HP and 1,3-PD by a newly isolated Lactobacillus renter! strain during whole cell biotransformation of glycerol [J]. Journal of Cleaner Production, 2019, 226 (432-42); (2) establishment of the multi-strain co-culture system for efficiently co-producing 3-HP and 1,3-PD mixing the resting cells of the L. re/tier! and the resting cells of the genetically engineered E. coil prepared above to obtain the multi-strain co-culture system In the system, the L. renter! has a mass fraction of 25% to 75% and the genetically engineered Li coil has a mass fraction of 25% to 75%.

The present invention also provides use of the above-mentioned multi-strain co-culture system in the efficient co-production of 3-HP and 1,3-PD by metabolizing glycerol.

Compared with the prior art, the present invention has the following beneficial effects.

In the present invention, a multi-strain co-culture strategy is used to avoid the inhibition of 3-HPA (a toxic intermediate metabolite) in the single-strain conversion of L. renter!. By integrating genetically engineered bacteria that can efficiently convert 3-HPA into 3-HP and/or 1,3-PD, 6 two-strain and three-strain co-culture systems are established to alleviate the inhibition of the intermediate metabolite.

In the present invention, UTR engineering modification is conducted on the genetically engineered E. col! co-expressing SSADH and 1,3-PD oxidoreductase in the optimal co-culture system, such that SSADH and 1,3-PD oxidoreductase are efficiently balanced and expressed, the problem of NAD+ and NADH supply during the conversion of 3-HPA into 3-HP and 1,3-PD is solved, and thus the generically engineered E. coil itself can realize the recycling of NAD+ and NADEL The present invention also develops a two-step method adapted to the above-mentioned optimal conversion system and a feed-batch bioconversion technology to further solve the problems of intermediate product inhibition and NAD-and NADH supply existing in the prior art, such that a reaction can continuously and efficiently proceed and a final total yield of 3-HP and 1,3-PD can reach 214.39 g/L, which is the highest level so far and has promising application prospects and practical significance.

Brief Description of the Drawings

FIG. 1 shows results of the production capacity comparison of the different co-culture systems (CS) according to the present invention.

FIG. 2 shows results of the conversion condition optimization for a two-strain co-culture system CS-2.

FIG. 3 shows comparison results of two-strain co-culture systems CS-2 with different UTR modifications.

FIG. 4 shows production capacities of the optimal two-strain co-culture system CS-2-V3 under different glycerol loads.

FIG. 5 shows yields of one-step and two-step conversion techniques at different glycerol concentrations.

FIG. 6 shows results of production using two feed-batch bioconversion technologies.

FIG. 7 shows the production capacity comparison of single-strain, two-strain, and three-strain conversion systems.

Detailed Description of the Embodiments

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the protection scope of the present invention is not limited thereto.

Example 1: Construction of genetically engineered E. cal: (1)E. coil expressing SSADH (E. coil BL21/pANY-GabD4, short for E. coil GabD4): According to a sequence of the gene GabD4 encoding SSADH in Cupriavidus necator and the characteristics of gene elements on the vector pANY1, the following primers were designed using Oli go7. 0 software: PA N Y-F: 5'-atgtatatctccttcttaaagt-3'; PAN Y-R: 5'-cctccatgggagctectg-3'; Gab D4-F 1: 5'-taactttaagaaggagatatacatatgtaccaagatctggcactgt-3'; and Gab D4-R1: 5'-tgcaggagcmccatggaggttacgcttgggtgatgaact-3'.

The primer pair of PANY-F and PANY-R was used to amplify a pANY1 vector backbone (excluding 6 x His tag and ccarB expression cassette), wherein the following PCR procedure was conducted to obtain the pANY1 vector backbone: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s; annealing at 55°C for 10 s; extension at 72°C for 30 s; and stopping extension at 72°C for 5 min, with 32 cycles.

The primer pair of GabD4-F I and GabD4-R I was used to amplify a gabD4 gene fragment with homologous arms, wherein the following PCR procedure was conducted to obtain the gabD4 gene: pre-denaturation at 98°C for 1 mm; denaturation at 98°C for 10 s; annealing at 55°C for 10 s; extension at 72°C for 30 s; and stopping extension at 72°C for 5 min, with 32 cycles.

The above two gene fragments were recombined and ligated according to the instructions of a seamless cloning kit. Specifically, the pANY1 vector backbone and the gabD4 gene were mixed in a ratio of L3 (mol/mol), a same volume of 2 x MultiF Seamless Assembly Mix was added, and a resulting mixture was subjected to ligation at 50°C for 30 min. A resulting recombinant vector was transformed into competent E.coli BL21 by heat shock. Specifically, a ligation product was added to competent cells, and a resulting mixture was thoroughly mixed, placed on ice for 30 mm, subjected to heat shock at 42°C for 60 s, and then cooled on ice for 2 min; and 900 pi of an LB medium was added, and a resulting mixture was incubated at 37°C for 1 h and then coated on a plate with 50 pg/mL kanamycin. A plasmid small extraction kit was used to extract the plasmid to verify whether its size was correct. Finally, the plasmid was sent to GENEWIZ for gene sequencing, and a result showed that the recombinant engineered strain was successfully screened, which was named E.coli BL21/ pANY-gabD4, short for E. colt GabD4.

(2) E. colt expressing 1,3-PD oxidoreductase (E. colt BL21/pANY-pch40, short for E. colt PduQ): The E. coil BL21/pANY-pduQ was constructed using a sequence of the gene PduQ encoding 1,3-PD oxidoreductase in L. reuteri and the vector pANY1. The method was basically the same as in step (1) except that the following primers were used: primers: PduQ-F 1: 5'-taactttaagaaggagatatacatatggaaaaarnagtatgccaac-3' and PduQ-R1 5'-tgcaggagcmccatggaggttaacgaattattgettcgtaaat-3'.

(3) E. coil co-expressing SSADH and 1,3-PD oxidoreductase (E. coil BL211pANY-gabl)4-pcluO, short for E. colt GabD4-PduQ): The following primers were designed using the vectors pANY-GabD4 and pANY-PduQ obtained in steps (1) and (2): GabD4-F2: 5'-cctccatgggagacctg-3'; GabD4-R2: 5'-ttacgcttgggtgatgaactt-3'; PduQ-F2: 5'-agttcatcacccaagcgtaatggccttttgctgg-3'; and PduQ-R2: 5'-tgcaggagctcccatggaggttaacgaattattgcttc-3'.

The E. colt co-expressing S S A DH and 1,3-PD oxidoreductase (E. colt BL21/pANY-GabD4-PduQ) was constructed by a method basically the same as in step (1).

Example 2:

(1) Preparation of resting cells: The L. reuteri FXZ014 was statically incubated overnight at 37°C in an MRS medium (10 g/L tryptone, 10 g/L beef extract, 5 g/L yeast powder, 2 g/L dipotassium phosphate (DKP), S g/L sodium acetate, 2 g/L ammonium citrate dibasic, 0.1 g/L magnesium sulfate, 0.15 g/L manganese sulfate, 1 g/L Tween 80, and 20 g/L glucose) for activation, and then subjected to expanded cultivation at 37°C for 12 h under anaerobic conditions in an MRS medium with 40 mN1 glycerol; a resulting bacterial solution was subjected to refrigerated centrifugation at 8,000 rpm and 4°C for 5 min, and a resulting supernatant was discarded; and finally, resulting cells were washed with a 0.1 M potassium phosphate buffer (pH 7.0) and centrifuged to obtain resting cells of the L. renter' FXZ014 for later use.

The three strains of E. colt GabD4, E. colt PduQ, and E. colt GabD4-PduQ were incubated overnight in an LB medium (10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast powder) for activation, and then subjected to expanded cultivation at 37°C and 220 rpm with an inoculation amount of 1% (v/v); when OD600 was 0.4 to 0.6, IPTG was added at a final concentration of 0.5 mNI, and a resulting mixture was incubated overnight at 25°C and 150 rpm to induce the protein expression; then a resulting bacterial solution was subjected to refrigerated centrifugation at 8,000 rpm and 4°C for 5 min, and a resulting supernatant was discarded; and finally, resulting cells were washed with a 0.1 M potassium phosphate buffer (pH 7.0) and then centrifuged to obtain resting cells of the E. coil GabD4, E. co/i PduQ, and E. coil GabD4-PduQ for later use.

(2) Measurement and conversion of resting cell dry weight: Strains activated overnight were inoculated respectively into 5 conical flasks each with 50 mL of a corresponding medium at an inoculation amount of 1%, and cultivated under suitable conditions. A sample was collected from each of the conical flasks every 3 h for OD600 determination, and a result was recorded; and then the sample was centrifuged to collect bacterial cells, and the bacterial cells were washed twice with a 0.1 M potassium phosphate buffer (pH 7.0) and then dried in an oven until a cell weight remained unchanged. Finally, 5 different 0D600 values and corresponding CDW values were obtained. An approximate relationship between 0D600 and CDW was obtained by a linear fitting tool: 1 OD600 0.34 g/L CDW.

Example 3: Establishment and screening of multi-strain co-culture systems In this example, multi-strain co-culture systems added with different bacteria were investigated for the co-production of 3-HP and 1,3-PD by metabolizing glycerol, thereby screening out the optimal multi-strain co-culture system. Different types and contents of bacteria were mixed to obtain the multi-strain co-culture systems in the experimental groups 1 to 6. Specific formulas were shown in Table I. Table 1 Formulas of the multi-strain co-culture systems Experimental group Formula 1 50% L. rotten FXZ014 + 25% Li coil GabD4 + 25% E. coil PduQ: 2 50% L. renteri FXZO 14 + 50% E. coil GabD4-PduQ 1 75% L. renter, FXZ014 + 12.5% Li coil GabD4 + 12.5% E. coil PduQ 4 75% L. renteri FXZO 14 + 25% E. coil GabD4-PduQ 25% L. reuteri FXZ014 + 37.5% E. coil GabD4 + 37.5% E. coil PduQ 6 25% L. renter' FXZ014 + 75% E. coil GabD4-PduQ The multi-strain co-culture systems (with CDW of 15 g/L) of different formulas in the experimental groups 1 to 6 were used separately to convert a 30 g/L glycerol solution, and 4 h later, samples were taken to determine the concentrations of 3-HP and 1,3-PD by high-performance liquid chromatography (HPLC) under the following conditions: Aminex HPX-87H (300 z 7.8 mm) chromatographic column, refractive index detector (RID), flow rate. 0 6 mL/min, and column temperature: 65°C. Determination results were shown in FIG. 1. It can be seen from FIG. 1 that, under the same conditions, total yields of 3-HP and 1,3-PD in the two-strain co-culture systems (CS-2, CS-4, and CS-6) were significantly higher than that in the three-strain co-culture systems (CS-1, CS-3, and CS-5), wherein yields of 3-HP and 1,3-PD in the co-culture system CS-2 were the highest, reaching 14.73 g/L and 10.38 g/L, respectively.

Example 4: Condition optimization for the two-strain co-culture system CS-2 In this example, the effects of different CDWs (20 g/L, 25 g/L, and 30 g/L), substrate concentrations (40 g/L, 50 g/L, and 60 g/L), and conversion time (3 h, 4 h, and 5 h) on the co-culture system CS-2 were investigated to further optimize the co-culture system CS-2 obtained in Example 3.

Test results were shown in FIG. 2. The results showed that, before 4 h, yields of 3-1-1P and 1,3-PD were significantly increased under all conditions, and after 4 h, the yields hardly changed; and when a CDW was 20 g/L and a glycerol concentration was 60 g/L, a total yield of the co-culture system reached the maximum value of 45.15 g/L, with 3-HP and 1,3-PD concentrations of 26.33 g/L and 18.82 g/L, respectively.

Example 5: Optimization for the two-strain co-culture system by UTR engineering (1) Establishment and screening of a UTR-modified two-strain mixed system: In this example, 5'-UTR engineered strains with different GabD4 expression levels were used instead of E. coil GabD4-PduQ to further optimize the two-strain co-culture system CS-2 screened out in Example 3. The 5 UTR engineered strains were E. con V1, E. coil V2, E. coil V3, E. coil V4, and E. con V5, respectively, which were obtained through UTR engineering and genetic engineering and had GabD4 expression levels from low to high.

Resting cells were prepared with reference to Example 2. L. renter' FXZ014 resting cells were mixed with each of.E. coil V1, E. coil V2, E. coil V3, E. coil V4, and E. coil V5 resting cells at a ratio of 11 to obtain the following systems: CS-2-V1, CS-2-V2, CS-2-V3, CS-2-V4, and CS-2-V5. A test was conducted according to the optimal conditions in Example 4: CDW: 20 g/L, glycerol concentration: 60 g/L, pH: 8.5, and conversion time: 4 h. Test results were shown in FIG. 3. Compared with the two-strain system CS-2, all two-strain systems undergoing UTR modification led to higher yields (FIG. 3a), wherein the two-strain system CS-2-V3 with E. coil V3 had the best performance, which almost completely consumed the 60 g/L glycerol (FIG. 3c), produced the least intermediate metabolite 3-1-IPA (FIG. 3b), and led to final 3-HP and 1,3-PD yields of 32.37 and 22.64 g/L, respectively.

(2) Yield comparison under high substrate load: In this step, the performance of the optimal two-strain system CS-2-V3 obtained in step (1) under a high glycerol load was investigated, and the capacities of a one-step method and a two-step method to co-produce 3-HIP and 1,3-PD by converting glycerol under a high glycerol load were compared. Generally, a high substrate concentration was a prerequisite for high yield, and the above-mentioned optimal two-strain system CS-2-V3 could almost completely consume the 60 g/L glycerol. Therefore, a high-concentration substrate conversion test for the two-strain system CS-2-V3 was conducted under the following conditions: high glycerol concentrations: 70 g/L, 85 g/L, 100 g/L, and 120 g/L, pH: 8.5, CDW: 20 WL, and temperature: 30°C.

Test results were shown in FIG. 4. It can be seen that, with the increase in the high glycerol load, yields of 3-HP and 1,3-PD increased (FIG. 4a), but the glycerol residue was also increased (FIG. 4c); under high glycerol loads, the intermediate metabolite 3-HPA accumulated with no significant difference (FIG. 4b); and pH measurement results showed that the pH gradually decreased with the reaction progress, and when the pH decreased to 6, the reaction almost stopped (FIG. 4d).

Example 6: One-step and two-step bioconversion One-step method: the two-strain mixed system CS-2-V3 was used to conduct conversion for 4 h under the following conditions: high glycerol concentrations: 120 g/L, 140 g/L, and 160 g/L, CDW: 20 g/L, pH: 8.5, temperature: 30°C, and rotational speed: 180 rpm; and then a concentration of each component was determined.

Two-step method: a reaction solution obtained from the one-step conversion was collected, and a pH was adjusted to 8.5; and a 20 g/L fresh resting cell solution (50% L. reuteri FXZ014 + 50% E. call V3) were added to allow a reaction for 4 h, and then a concentration of each component was determined.

Test results were shown in FIG. S. It can be seen that the two-strain mixed system CS-2-V3 failed to fully utilize glycerol at 120 g/L and higher concentrations through the one-step conversion, and the higher the substrate concentration, the larger the substrate inhibition effect and the intermediate metabolite inhibition effect; and the two-step conversion based on the one-step conversion could fully utilize the 120 g/L glycerol, with final 3-HP and 1,3-PD concentrations of 69.36 g/L and 47.58 g/L, wherein the intermediate 3-HPA was not detected. When the two-step method was used to convert 140 g/L glycerol, the yield was higher than that when 120 g/L glycerol was used, but 12.56 g/L glycerol failed to be converted. When 160 g/L glycerol was used, the yield was even lower than that when 140 g/L substrate was used due to inhibition effects of the substrate and intermediate.

Example 7: Continuous feed-batch bioconversion In this example, in order to completely eliminate the inhibition effects of the substrate and intermediate, two feed-batch bioconversion technologies were developed for the two-strain mixed system CS-2-V3, which greatly improved the capacity to co-produce 3-HP and 1,3-PD.

(1) Continuous feed of glycerol Initial conditions were as follows: CDW: 20 g/L, glycerol concentration: 20 g/L, pH: 8.5, temperature: 30°C, and rotational speed: 180 rpm. 2 h after conversion, samples were collected to analyze components, then 20 g/L glycerol was fed, and a pH was adjusted to 8.5; and the above operation was repeated every 2 h until a final yield remained unchanged. Test results were shown in FIG. 6a. The strategy of feeding low-concentration substrate in batches was adopted, such that the reaction continued for 18 h and 94.56 g/L of 3-HP and 64.13 g/L of 1,3-PD were produced.

(2) Continuous feed of glycerol and resting cells Initial conditions were as follows: CDW: 10 g/L, glycerol concentration: 20 g/L, pH: 8.5, temperature: 30°C, and rotational speed: 180 rpm. 2 h after conversion, samples were collected to analyze components, then 20 g/L glycerol and resting cells with a CDW of 3 g/L were fed, and a pH was adjusted to 8.5; and the above operation was repeated every 2 h until a final yield remained unchanged.

Test results were shown in FIG. 6b. It can be seen that, at a low initial resting cell concentration and a low initial substrate concentration, the intermediate metabolite inhibition effect and the low temperature inhibition effect were almost eliminated; freshly added resting cells also made the reaction last longer (24 h); and after the 24 h of reaction, 240 g/L glycerol was consumed to produce 214.39 g/L of 3-1-IP and 1,3-PD in total, with a 3-HP yield of 125.93 g/L and a 1,3-PD yield of 88.46 g/L. The 3-HP and 1,3-PD production capacity was as high as 8.93 g/L/h, which is the highest level so far.

Comparative Example 1: Comparison of glycerol conversion yields of the single-strain, two-strain, and three-strain conversion systems In this example, in order to investigate the differences in glycerol conversion yields of the single-strain, two-strain, and three-strain conversion systems, the single-strain (L. renter' FXZ014 20 g/L), two-strain (L. reuleri FXZ014 10 g/L and E. coil BL21/pANY-GabD4-PduQ 10 g/L), and three-strain (L. renter, FXZ014 10 g/L, AI coil BL21/pANY-GabD4 5 g/L, and E. coil BL21/pANY-PduQ 5 g/L) conversion systems were separately set. The systems separately reacted with 40 g/L glycerol at 30°C and 180 rpm for 6 h, and then concentrations of 3-HP, 1,3-PD, and glycerol residue were determined.

As shown in FIG. 7, in the conversion of the single-strain system, a large amount of the intermediate metabolite 3-HPA accumulated, and only 8.9 g/L of 3-HP and 6.2 g/L of 1,3-PD were produced; and compared with the conversion of the single-strain system, the conversion strategy using the multi-strain mixed system significantly alleviated the intermediate inhibition effect and increased the yields of 3-HP and 1,3-PD, wherein the two-strain conversion strategy exhibited the best effect, producing 15.3 g/L of 3-HP and 11.9 g/L of 1,3-PD.

Comparative Example 2: Yield comparison of the one-step bioconversion, two-step bioconversion, and continuous feed-batch bioconversion In this example, different effects of the two-strain system CS-2-V3 when used by different conversion technologies for co-producing 3-HP and 1,3-PD were investigated. The one-step conversion and the two-step conversion were conducted as in the above example, and the glycerol concentration increased until the yield no longer increased. Results were shown in FIG. 8. It can be seen that the general one-step conversion led to the lowest total yield, which was only 85.2 g/L; the two-step conversion developed in the present invention increased the total yield by 36%; and the two feed-batch bioconversion technologies developed in the present invention exhibited unparalleled advantages and led to a total yield almost twice that of the general one-step conversion, reaching 214.39 g/L, which is also the optimal level so far.

The above examples are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvement, substitution, or modification made by those skilled in the art without departing from the essence of the present invention should fall within the protection scope of the present invention.

Claims (9)

1. Claims What is claimed is: 1. A multi-strain co-culture system, characterized by comprising Lactobacillus it. uteri (L. reuleri) and genetically engineered Eccherichia co/i (E. co/i), wherein in the system, the L. renter' has a mass fraction of 25% to 75% and the genetically engineered E con has a mass fraction of 25% to 75%.
2. 2. The multi-strain co-culture system according to claim 1, characterized in that the genetically engineered E cot/ is one or more from the group consisting of E co/i expressing succinic semialdehyde dehydrogenase (SSADH) co/i GabD4), K co/i expressing 1,3-propanediol (1,3-PD) oxidoreductase co/i PduQ), and E co/i co-expressing the SSADH and the 1,3-PD oxidoreductase (E co/i GabD4-PduQ).
3. 3. The multi-strain co-culture system according to claim 1, characterized in that the bacteria in the system have a total concentration of 10 g/L to 30 g/L based on cell dry weight (CDW).
4. 4. The multi-strain co-culture system according to claim 1, characterized in that in the system, the L. renter' has a mass fraction of 50% and the genetically engineered E co/i has a mass fraction of 50%.
5. 5. The multi-strain co-culture system according to any one of claims 1 to 4, characterized in that in the system, the genetically engineered E. co/i is E. colt GabD4-PduQ, and in the system, the L. rented has a mass fraction of 50%, the E. coil GabD4-PduQ has a mass fraction of 50%, and the bacteria have a total concentration of 20 g/L based on CDW.
6. 6. An establishment method of the multi-strain co-culture system according to claim 1, characterized by comprising: preparing resting cells of L. renteri FXZ014 and the genetically engineered E co/i, separately, and mixing the resting cells in a predetermined ratio to obtain the multi-strain co-culture system.
7. 7. The establishment method of the multi-strain co-culture system according to claim 6, characterized in that in the multi-strain co-culture system, the L. renter, FXZ014 has a mass fraction of 25% to 75% and the genetically engineered E. coil has a mass fraction of 25% to 75%.
8. 8. Use of the multi-strain co-culture system according to claim 1 in the co-production of 3-hydroxypropionic acid (3-HP) and 1,3-PD.
9. 9. The use according to claim 8, characterized in that the genetically engineered bacteria achieve the co-production of 3-HP and 1,3-PD by metabolizing glycerol.