The invention relates to a method for screening crude lactose-resistant escherichia coli through adaptive laboratory evolution and a method for screening crude lactose-resistant escherichia coli in 2apos; application in fucosyllactose production

Abstract

The invention discloses a construction method and application of a 2 '-fucosyllactose production strain tolerant to high-ash-content crude lactose, and belongs to the technical field of bioengineering. In order to solve the problems that the existing 2 '-fucosyllactose fermentation production depends on expensive refined lactose, and ash substances in low-cost crude lactose can inhibit product synthesis, escherichia coli with 2'-fucosyllactose production capacity is used as an original strain, an M9 basic culture medium is used as a subculture medium, and the 2 '-fucosyllactose fermentation production method is used for producing 2'-fucosyllactose. An adaptive laboratory evolution (ALE) strategy of gradient increase of the ratio of the coarse lactose is adopted, and an evolved strain capable of efficiently utilizing the high-ash-content coarse lactose is obtained through multiple rounds of continuous passage domestication and directional screening. When the strain constructed by the invention is fermented by using crude lactose, the yield of 2 '-fucosyllactose can reach 140 g / L, which is equivalent to that by using refined lactose, and the strain grows stably. The method does not need complex gene modification, is simple and convenient to operate, remarkably reduces the industrial production cost, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a method for producing 2'-fucosylated lactose (2'-FL) Escherichia coli through adaptive laboratory evolution screening to produce crude lactose-tolerant Escherichia coli, which belongs to the field of synthetic biology. Background Technology

[0002] 2'-Fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk, accounting for approximately 30% of the total oligosaccharides in human milk. Studies have shown that 2-FL has multiple important physiological functions, including regulating the gut microbiota of infants, inhibiting pathogen adhesion, enhancing immune function, and promoting brain development. It is an important functional additive in infant formula, and market demand for it is growing.

[0003] Lactose is an indispensable precursor in the biosynthetic pathway of 2-FL. A fucose molecule is linked to the galactose terminus of the lactose molecule via an α-1,2 glycosidic bond, allowing the complete lactose molecule to directly form the "skeleton" structure of 2-FL. Therefore, lactose plays a crucial role in the 2-FL synthesis pathway, and its quality and cost directly affect the production efficiency and economic benefits of 2-FL.

[0004] Currently, the industrial production of 2-FL mainly employs microbial fermentation. During fermentation, high-purity refined lactose is typically used as a precursor. However, high-purity refined lactose is expensive, accounting for a significant portion of production costs. To reduce production costs, industry has attempted to use low-cost crude lactose (such as whey permeate) as a substitute for refined lactose. However, crude lactose usually contains a high amount of ash (approximately 0.26 g / 100 g). Ash is the inorganic mineral residue left after the substance is burned, mainly consisting of a mixture of various inorganic elements such as metal oxides, phosphates, sulfates, silicates, and chlorides.

[0005] Studies have found that the ash in crude lactose has a significant negative impact on the producing strains. Even in trace amounts, certain metal ions in the ash can be physiologically toxic to the bacteria; furthermore, for non-halophilic Escherichia coli, these salts can hinder transmembrane transport and cause osmotic imbalance, thus affecting the normal metabolic function of the bacteria. Experiments show that direct fermentation using crude lactose leads to a 28.5%–64.2% decrease in 2-FL yield, severely impacting production efficiency.

[0006] Therefore, how to obtain production strains that can tolerate the ash content in crude lactose, so that they can maintain a fermentation yield comparable to that of refined lactose when using low-cost crude lactose, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] 1. Purpose of the invention

[0008] To address the problem that existing technologies for 2'-fucosylated lactose (2-FL) fermentation production rely on high-purity refined lactose, resulting in high raw material costs, the present invention aims to provide a method for constructing a 2'-fucosylated lactose-producing strain tolerant to high-ash crude lactose. The evolved strain obtained through this method can overcome the inhibitory effect of ash on cell metabolism when using low-cost, high-ash crude lactose (such as whey permeate) as a substrate, maintaining fermentation yields comparable to those using refined lactose, thereby significantly reducing industrial production costs.

[0009] 2. Technical Solution

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention provides a method for constructing a 2'-fucosylated lactose-producing strain tolerant to high-ash crude lactose, the method comprising the following steps:

[0012] (1) Preparation of starting strain

[0013] Escherichia coli ZNT01, which has a high production capacity of 2'-fucosylated lactose, was selected as the starting strain.

[0014] (2) Adaptive laboratory evolution

[0015] Using M9 basal medium as the basic culture medium for subculture, the starting strain was subjected to multiple rounds of continuous subculture domestication using an adaptive laboratory evolution (ALE) strategy that gradually increases the proportion of crude lactose.

[0016] The cultivation conditions for the acclimatization process were controlled as follows: temperature 35~38℃, rotation speed 180~240 rpm, cultivation time per generation 10~14 h, and inoculum size 0.5%~5% (v / v). The amount of lactose stock solution added was 1%~5% (v / v) of the culture volume.

[0017] The specific acclimatization gradient and round settings are as follows: First round of domestication: Set the ratio of refined lactose to crude lactose to (3~5):1, and pass it on for 4~6 generations; Second round of domestication: Set the ratio of refined lactose to crude lactose to (1.5~2.5):(2.5~3.5) (preferably 2:3), and pass it on for 5~7 generations; Third round of domestication: Set the ratio of refined lactose to crude lactose to 1:(3~5), and pass it on for 4~6 generations; Fourth round of domestication: Use only crude lactose (ratio 0:1) and pass it on for 4 to 6 generations.

[0018] (3) Screening of dominant strains

[0019] After each round of domestication, single colonies were isolated by streak plating of the domesticated bacterial solution. After incubation at 35–38°C for 10–14 h, single colonies were picked. Using the fermentation medium as the selection medium and crude lactose as the sole fermentation substrate, the cultures were incubated at 35–38°C and 180–240 rpm for 46–50 h. The yield of 2'-fucosylated lactose in the fermentation broth was measured, and the dominant strain with the highest yield was selected as the starting strain for the next round of domestication.

[0020] (4) Obtain the target strain

[0021] Repeat steps (2) and (3) until the selected strain can fully tolerate 100% crude lactose and the yield meets expectations, thus obtaining a 2'-fucosylated lactose production strain that is tolerant to high ash crude lactose.

[0022] 3. Beneficial effects

[0023] Compared with the prior art, the present invention has the following significant advantages:

[0024] a) Significantly reduced raw material costs: The evolved strain obtained by this invention breaks the dependence of 2-FL production on high-purity refined lactose and can directly use inexpensive crude lactose for fermentation production, which greatly reduces raw material costs and enhances the market competitiveness of the product.

[0025] b) Yield not inhibited by ash: The strain constructed in this invention exhibits excellent tolerance to the inhibitory effects of ash substances (such as inorganic salts and metal oxides at a content of 0.26 g / 100 g) present in crude lactose on bacterial cells. Experimental data show that the evolved strain can achieve a 2-FL yield of 140 g / L when fermenting with crude lactose, comparable to the yield of the control group using refined lactose, thus solving the technical problems of low crude lactose utilization and inhibited product synthesis.

[0026] c) Stable growth and genetic performance of the strain: Through multiple rounds and long-term gradient pressure acclimatization, the obtained strains exhibit stable genetic traits. During fermentation, the cell growth curve (OD600) remained stable, indicating that the strains had successfully adapted to the high osmotic pressure and complex ionic environment;

[0027] d) The method is simple, efficient, and highly versatile: The ALE strategy employed in this invention is easy to operate and requires no complex genetic engineering modifications to the strains. Through a cycle of "pressure selection - spontaneous mutation - directed selection," the strains naturally evolve to achieve optimal metabolic flux. Furthermore, this strategy is universally applicable and can be extended to substrate adaptation modifications of other lactose oligosaccharide-producing strains, demonstrating broad industrial application prospects. Attached Figure Description

[0028] Figure 1 Comparison of cell OD600 during the fermentation process of crude lactose and refined lactose in a 5L fermenter;

[0029] Figure 2 Comparison of 2-FL yield during the fermentation process of crude lactose and refined lactose in a 5L fermenter;

[0030] Figure 3 Comparison of OD600 and 2-FL yield under different combinations of lactose and ash substances in shake flask fermentation;

[0031] Figure 4 Comparison of OD600 and 2-FL yields of shake-flask fermentation of the first round of trained dominant strains;

[0032] Figure 5 Comparison of OD600 and 2-FL yield in shake-flask fermentation of the dominant strains in the second round of domestication;

[0033] Figure 6 Comparison of OD600 and 2-FL yield in shake-flask fermentation of the dominant strains in the third round of domestication;

[0034] Figure 7 Comparison of OD600 and 2-FL yield in shake-flask fermentation of the dominant strains in the fourth round of domestication;

[0035] Figure 8 Comparison of 2-FL yields of strains T4-5 fermented with different amounts of lactose in 5L;

[0036] Figure 9 Comparison of OD600 of strain T4-5 fermented with different amounts of lactose in 5L. Detailed Implementation

[0037] Implementation Case 1: Comparison of Fermentation Yields of Crude Lactose Strains and Refined Lactose

[0038] In the fermentation production of 2-FL, to reduce raw material costs, this embodiment attempts to use low-cost crude lactose (containing ash) instead of expensive refined lactose. First, ZNT01 was used as the fermentation strain, and parallel verification was conducted in a 5L fermenter. Except for the source of lactose (one group used refined lactose, and the other used crude lactose), all other culture conditions were controlled within the same range: temperature controlled at 34–36°C, pH maintained at 6.7–6.9, tank pressure 0.15–0.25 MPa, dissolved oxygen maintained at 30%–40%, and lactose was pumped into the fermentation broth via a feedstock method. Samples were taken and monitored every 3–5 hours during fermentation. The monitoring results are as follows: Figure 1 The results showed that the OD600 growth curves of the two groups of strains were almost identical, indicating that the utilization of crude lactose did not significantly affect the growth of the cells. However, as... Figure 2 The yield data for 2-FL showed significant differences: the strain utilizing crude lactose consistently produced lower yields than the strain utilizing refined lactose. During fermentation periods of 72–76 h, the refined lactose group achieved a yield of 140 g / L, while the crude lactose group only yielded 100 g / L, a decrease of 28.5%. This indicates that while the use of crude lactose supported cell growth, it significantly inhibited 2-FL synthesis.

[0039] To investigate the reasons for the yield decline, the composition of crude lactose was analyzed, revealing approximately 0.26 g / 100g of ash. It was speculated that even trace amounts of metal oxides or salt mixtures in the ash could potentially have physiological toxicity to *E. coli*, or lead to impaired transmembrane transport and osmotic imbalance. To verify this hypothesis, a shake-flask verification experiment was further conducted in this embodiment.

[0040] The experiment used ZNT01 as the starting strain and cultured it in fermentation medium. The fermentation medium formula is as follows: glycerol 15-25 g / L, yeast extract 3-8 g / L, peptone 8-12 g / L, Na2HPO4·12H2O 15-20 g / L, NH4HPO4 0.5-1.5 g / L, K2HPO4 2-4 g / L, NH4Cl 1-3 g / L, MgSO4·7H2O 1-2 g / L, trisodium citrate 1-3 g / L, vitamin B1 5-15 mg / L; and trace elements (FeCl3·6H2O 20-30 mg / L, CaCl2·2H2O 1-4 mg / L, ZnCl2 2-4 mg / L, CuSO4·5H2O 1-3 mg / L, MnSO4·H2O 2-3 mg / L, Na2MoO4·2H2O 2-3 mg / L, H3BO3). (0.5~1.0 mg / L).

[0041] The experiment was conducted in four parallel groups, incubated at 35–38℃ and 180–220 rpm for 46–50 h: Group A was supplemented with refined lactose; Group B was supplemented with crude lactose; Group C was supplemented with refined lactose and ash (ash content calculated at 0.26 g / 100 g lactose); Group D was supplemented with only ash. Figure 3 As shown, the results after fermentation for 46-50 h were as follows: Group A (refined lactose) yielded 12 g / L; Group B (crude lactose) yielded 5.1 g / L, a decrease of 57.5% compared to Group A; Group C (refined lactose + ash) yielded 4.3 g / L, a decrease of 64.2% compared to Group A; and Group D yielded zero.

[0042] The experimental data above confirms that artificially adding ash to refined lactose leads to a significant decrease in yield, similar to the effect of using crude lactose directly. This indicates that ash in crude lactose is the main factor causing the reduced yield of ZNT01 strain 2-FL, and that this strain cannot effectively tolerate or utilize ash. Based on this conclusion, this invention plans to employ an adaptive laboratory evolution (ALE) strategy to screen for production strains that can tolerate high ash content and achieve efficient conversion of crude lactose.

[0043] Implementation Case 2: Adaptive Laboratory Evolutionary Screening for Lactose-Tolerant Strains (First Round of Domestication)

[0044] Adaptive Laboratory Evolution (ALE) is a commonly used irrational breeding technique in the modification of microbial fermentation production. This invention uses strain ZNT01 as the starting strain, employs crude lactose with high ash content as the selection pressure, and continuously subcultures the starting strain by controlling the ratio of refined lactose to crude lactose through gradient control. The specific process is as follows:

[0045] M9 basal medium (formulation: Na2HPO4·12H2O 17.1 g / L, KH2PO4 3 g / L, glucose 4 g / L, NH4Cl 1 g / L, NaCl 0.5 g / L, MgSO4 0.24 g / L, CaCl2 0.011 g / L) was used as the subculture medium, with crude lactose added as a screening pressure. The initial ratio of purified lactose to crude lactose was 4:1. 30 mL of M9 basal medium was added to each 500 mL round-bottom flask, and the mixed lactose stock solution was prepared to a concentration of 300 g / L. The culture was then autoclaved at 121°C for 20 min.

[0046] The initial bacterial strain was activated in LB shakers at 35–38°C and 180–220 rpm for 10–14 h. Then, it was inoculated into sterilized M9 basal medium at an inoculation volume of 0.5%–2%, with 2% mixed lactose stock solution added. The culture was then incubated at 35–38°C and 180–220 rpm for 10–14 h, designated as the first generation. After this first generation, the culture was transferred to fresh M9 basal medium, and the same volume of bacterial culture and mixed lactose was added. After incubation for 10–14 h, this was designated as the second generation, and the process was repeated. After five generations, the bacterial culture was streaked onto plates and activated at 35–38°C for 10–14 h. Single colonies were then selected for fermentation verification (relatively large and healthy colonies were chosen).

[0047] Nine single colonies were selected and named T1-1 to T1-9, with strain ZNT01 serving as the control. Groups T1-1 to T1-9 and ZNT01 used fermentation medium and crude lactose as the fermentation substrate, while group ZNT01 used fermentation medium and refined lactose. The cultures were incubated at 35–38℃ and 180–220 rpm on a shaker for 46–50 h. After fermentation, the 2-FL yield and OD600 of the strains were measured. Figure 4 The results showed that among all the passaged strains, strain T1-4 had the highest yield of 5.9 g / L, which was 40% higher than the control group ZNT01. However, it was still lower than the fermentation results of the strain using refined lactose. Therefore, strain T1-4 was selected as the initial strain for further passage.

[0048] Implementation Case 3: Adaptive Laboratory Evolutionary Screening for Crude Lactose-Tolerant Strains (Second Round of Domestication)

[0049] The ratio of refined lactose to crude lactose was adjusted to 2:3. M9 basal culture medium was added at a rate of 30 mL per 500 mL round-bottom bottle, and the mixed lactose stock solution was prepared to a concentration of 300 g / L. The mixture was then autoclaved at 121°C for 20 min.

[0050] After activating strains T1-4 in LB shakers at 35-38℃ and 180-220 rpm for 10-14 h, they were inoculated into sterilized M9 basic medium at an inoculation volume of 0.5%-2%, with 2% mixed lactose stock solution added simultaneously. The culture was then incubated at 35-38℃ and 180-220 rpm for 10-14 h, designated as the first generation. After the first generation, the culture was transferred to fresh M9 basic medium and incubated for 10-14 h, designated as the second generation. This process was repeated sequentially. After six generations, the bacterial culture was streaked onto plates and activated at 35-38℃ for 10-14 h. Single colonies were then selected for fermentation verification.

[0051] Eight single colonies were selected and named T2-1 to T2-8, with strain T1-4 serving as the control strain. Groups T2-1 to T2-8 and T1-4-1 used fermentation medium and crude lactose as the fermentation substrate, while group T1-4 used fermentation medium and refined lactose. The cultures were incubated at 35–38℃ and 180–220 rpm for 46–50 h before testing. Figure 5 The results showed that among all the passaged strains, strain T2-8 had the highest yield of 7.1 g / L, which was 26.7% higher than the control group T1-4-1. However, it was still lower than the fermentation results of group T1-4 using refined lactose. Therefore, strain T2-8 was selected as the initial strain for further passage.

[0052] Implementation Case 4: Adaptive Laboratory Evolutionary Screening for Lactose-Tolerant Strains (Third Round of Domestication)

[0053] The ratio of refined lactose to crude lactose was adjusted to 1:4. M9 basal culture medium was added at a rate of 30 mL per 500 mL round-bottom bottle, and the mixed lactose stock solution was prepared to a concentration of 300 g / L. The mixture was then autoclaved at 121°C for 20 min.

[0054] After activation of strain T2-8 in LB shake flasks, it was inoculated into sterilized M9 basic medium at an inoculum rate of 0.5%–2%, with 2% mixed lactose stock solution added simultaneously. The culture was incubated at 35–38°C and 180–220 rpm for 10–14 h as one generation, and this method was used for continuous subculturing. After 5 generations, the bacterial culture was streaked onto plates and activated at 35–38°C for 10–14 h. Single colonies were then picked for fermentation verification.

[0055] Eight single colonies were selected and named T3-1 to T3-8, with strain T2-8 serving as the control strain. Groups T3-1 to T3-8 and T2-8-1 used fermentation medium and crude lactose as the fermentation substrate, while group T2-8 used fermentation medium and refined lactose. The cultures were incubated at 35–38℃ and 180–220 rpm for 46–50 h before testing. Figure 6 The results showed that among all the passaged strains, strain T3-1 had the highest yield of 10.3 g / L, which was 49% higher than the control group T2-8-1. However, it was still lower than the fermentation results of group T2-8 using refined lactose. Therefore, strain T3-1 was selected as the initial strain for further passage.

[0056] Implementation Case 5: Adaptive Laboratory Evolutionary Screening for Lactose-Tolerant Strains (Fourth Round of Domestication)

[0057] All crude lactose was used (ratio 0:1). M9 basic culture medium was added at a rate of 30 mL per 500 mL round-bottom bottle, and crude lactose stock solution was prepared at 300 g / L. The mixture was then autoclaved at 121°C for 20 min.

[0058] After activating strain T3-1 using LB shakers, it was inoculated into sterilized M9 basal medium at an inoculum rate of 0.5%–2%, with 2% crude lactose stock solution added simultaneously. The culture was incubated at 35–38°C and 180–220 rpm for 10–14 h as one generation, and this method was used for continuous subculturing. After 5 generations, the bacterial culture was streaked onto plates and activated at 35–38°C for 10–14 h. Single colonies were then picked for fermentation verification.

[0059] Eight single colonies were selected and named T4-1 to T4-8, with strain T3-1 serving as the control. Groups T4-1 to T4-8 and T3-1-1 used fermentation medium and crude lactose as the fermentation substrate, while group T3-1 used fermentation medium and refined lactose. The cultures were incubated at 35–38℃ and 180–220 rpm for 46–50 h before testing. Figure 7 The results showed that strain T4-5 had the highest yield among all passaged strains, at 12.7 g / L, similar to the yield level using refined lactose. Subsequent small-scale trials in a 5L fermenter further verified that this strain was not affected by the ash content in crude lactose.

[0060] Implementation Case 6: Validation of 5L Fermentation Level of T4-5

[0061] Using strain T4-5 as the fermentation strain, fermentation was validated in a 5 L fermenter at a temperature of 35℃, pH 6.8, pressure 0.2, and dissolved oxygen maintained at 35℃. During the process, extrinsic lactose and neolactose were added separately, and samples were taken every 4 h to monitor fermentation OD600 and 2-FL yield.

[0062] Fermentation validation results from a 5 L fermenter showed that, Figure 8 and 9 The results showed that, compared with the control strain, the yield of the evolved strain T4-5 was unaffected by the use of the new lactose during fermentation, remaining at 140 g / L, and the OD remained stable. These results demonstrate that adaptive laboratory evolution significantly achieved efficient conversion of the production strain to the new lactose.

Claims

1. A method for constructing a 2'-fucosylated lactose-producing strain tolerant to high-ash crude lactose, characterized in that, Includes the following steps: (1) Preparation of starting strain: Escherichia coli with the ability to synthesize 2'-fucosylated lactose was selected as the starting strain; (2) Adaptive laboratory evolution: Inorganic salt medium was used as the basic culture medium for subculture, lactose was used as the carbon source, and a strategy of gradually increasing the proportion of crude lactose in the total lactose was adopted to carry out multiple rounds of continuous subculture domestication of the starting strain. (3) Screening of dominant strains: After each round of domestication, the bacterial solution was streaked to separate the bacteria, and single colonies were selected for fermentation verification using crude lactose. The strain with the highest 2'-fucosylated lactose production was selected as the starting strain for the next round of domestication. Repeat steps (2) and (3) until the target strain that can fully utilize crude lactose for production is obtained.

2. The construction method according to claim 1, characterized in that, The "gradual increase in the proportion of crude lactose to total lactose" mentioned in step (2) specifically includes the following four acclimatization rounds: First round: The mass ratio of refined lactose to crude lactose is 3~5:1; Second round: The mass ratio of refined lactose to crude lactose is 1.5~2.5:2.5~3.5; Third round: The mass ratio of refined lactose to crude lactose is 1:3~5; Round 4: Use only crude lactose in a 0:1 ratio.

3. The construction method according to claim 1 or 2, characterized in that, The culture conditions for continuous subculturing and domestication described in step (2) are: temperature 35~38℃, rotation speed 180~240 rpm, culture time per generation 10~14 h, and inoculum size 0.5%~5% (v / v).

4. The construction method according to claim 1, characterized in that, The inorganic salt culture medium mentioned in step (2) is M9 basic culture medium, whose components include: Na2HPO4·12H2O 17.1 g / L, KH2PO4 3 g / L, glucose 4 g / L, NH4Cl 1 g / L, NaCl 0.5 g / L, MgSO4 0.24 g / L, and CaCl2 0.011 g / L.

5. The construction method according to claim 1, characterized in that, The fermentation verification in step (3) uses a fermentation medium with the following component content ranges: glycerol 15~25 g / L, yeast extract 3~8 g / L, peptone 8~12 g / L, Na2HPO4·12H2O 15~20 g / L, NH4HPO4 0.5~1.5 g / L, K2HPO4 2~4 g / L, NH4Cl 1~3 g / L, MgSO4·7H2O 1~2 g / L, trisodium citrate 1~3 g / L, VB1 5~15 mg / L; and trace elements (FeCl3·6H2O 20~30 mg / L, CaCl2·2H2O 1~4 mg / L, ZnCl2 2~4 mg / L, CuSO4·5H2O 1~3 mg / L, MnSO4·H2O 2~3 mg / L, Na2MoO4·2H2O 2~3 mg / L). mg / L, H3BO30.5~1.0 mg / L).

6. The construction method according to claim 1, characterized in that, The crude lactose is an unrefined lactose product or whey permeate containing 0.2% or more ash by weight; the ash includes one or more of metal oxides, phosphates, sulfates or chlorides.

7. A strain of 2'-fucosylated lactose-producing bacteria tolerant to high-ash crude lactose, characterized in that, The production strain is obtained by constructing the method according to any one of claims 1-6.

8. The application of the 2'-fucosylated lactose-producing strain tolerant to high-ash crude lactose as described in claim 7 in the fermentation production of 2'-fucosylated lactose.

9. The application according to claim 8, characterized in that, The fermentation substrate used in the fermentation process is crude lactose or whey permeate, and no additional refined lactose needs to be added.

Images