An E. coli gene-knockout vector was constructed using pET28a (+) vector (Sangon, Shanghai, China) and cultured in Luria Broth (LB) medium at 37°C. E. coli DH5α (Vazyme Biotech, Nanjing, China) cells were used as hosts for gene cloning in Luria Broth (LB) medium at 37°C. The L-lysine-producing strain E. coli CGMCC 1.366 strain (from our laboratory) was used as the host to carry out gene cloning and culture in fermentation medium for β-alanine production. The fermentation formula for producing β-alanine by E. coli is as follows: 15.1 g Na₂HPO₄ 12H₂O, 3 g KH₂PO₄, 1 g NH₄CI, 0.5 g NaCl, 13.21 g (NH₄)₂SO₄, 20 g glucose, 10.0 g FeSO₄·7H₂O, 1.0 g CuSO₄·5H₂O, 0.5 g MnSO₄·4H₂O, 2.3 g ZnSO₄·7H₂O, 0.1 g (NH₄)Mo₇O₂₄, 0.2 g, Na₂B₄O₇·10H₂O, 2.0 g CaCl₂ and 25 mg, L-lysine. The pH was adjusted to 7 with 100 g L⁻¹ NaHCO₃. The primers were synthesized by Sangon Biotech (Shanghai, China). All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States). The enzyme and plasmid preparation kits for restriction enzymes were provided by Vazyme (Nanjing, China). All the strains and plasmids used in this study are listed in Table 1. All the primers used in this study are listed in Supplementary Table S1.

The genome of E. coli was used as the template to search for the gene encoding aspartate kinase lysC (WP_203077836.1) homologous protein on the NCBI official website to determine its gene length and sequence. The aspartokinase gene (Dookeran and Nielsen, 2021) in the L-lysine-producing E. coli pathway was knocked out using the RED homologous recombination experiment (Chao and Hu, 2022). The upper and lower homology arms (lysC1 and lysC2) were amplified by PCR using the upper and lower homology arm primers, lysC1-F/R and lysC2-F/R, respectively, designed using the CE design software. The FRT-Kan-FRT sequence in plasmid pKD13 was amplified by PCR using the upstream and downstream primers, FRT-Kan-FRT-F/R, of the FRT site fragment with overlapping homologous arms. The amplification conditions of the three fragments were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min using 30 cycles, and a final extension at 72°C for 5 min. Purification and recovery was performed using a gel recovery kit (Vazyme Biotech, Nanjing, China) and gene fragments were obtained, including a 500 bp fragment of lysC1, a 500 bp fragment of lysC2, and a 1,324 bp fragment of FRT-Kan-FRT. The plasmid pET28a (+) was double-digested with restriction enzymes Nde I and Hind III (Thermo Fisher Scientific, United States), purified, and recovered (Valente et al., 2021). A one-step cloning kit was used for seamless cloning (Vazyme Biotech, Nanjing, China). The concentrations of the target genes lysC1, lysC2, and FRT-Kan-FRT recovered from the gel and expression vector pET28a (+) recovered after double enzyme digestion purification were detected using a MD2000 microspectrophotometer (UVITECe, United Kingdom). The gene fragments were diluted so that the concentration of the three target genes was 10 ng/μL, and the concentration of linearized pET28a (+) vector was 100 ng/μL. The PCR was performed at 37°C for 30 min, and the fragments obtained by overlapping polymerase chain reaction were cloned into plasmid DH5α. Individual colonies growing on the transformed plate (50 μg/mL kanamycin) were cultured in LB liquid medium with corresponding resistance markers at 37°C and 200 r/min overnight on a shaker. The pET28a (+)-lysC1-FRT-Kan-FRT-lysC2 plasmid was extracted and used as a template to amplify the knockout frame FRT-CKan-FRT with Ck-F/R upstream and downstream primers. The length of the knockout box (FRT-CKan-FRT fragment) was 2,324 bp.

E. coli CGMCC 1.366 competent cells (Yang et al., 2022) were prepared in advance. Single colonies were inoculated in 50 mL liquid LB, and the cell density in the bacterial solution was controlled at OD₆₀₀ = 0.2 by shaking culture at 37°C. The temperature-sensitive plasmid pKD46 containing arabinose (Liao et al., 2018) induced recombinase was introduced into the strain, and E. coli CGMCC 1.366 competent cells (prepared in a frozen state and stored at −80°C) were quickly taken out and placed on ice (0°C) for thawing (approximately 10–15 min). Ten microliters of pKD46 plasmid was gently added to competent cells to avoid the generation of bubbles. The bottom of the centrifuge tube was gently tapped with a finger for mixing and the tube was placed on ice for half an hour. The sample was heat shocked in a 42°C water bath for 90 s, followed by cooling on ice at 0°C for 2–3 min. Then, 900 μL of LB liquid medium was added to the centrifuge tube to recover the transformed cells. The pKD46 plasmid is temperature-sensitive; therefore, the recovery culture condition was 30°C at 200 rpm for 1 h. The bacterial solution was centrifuged at 5,000 r/min for 5 min, transferred to a sterile super-clean bench, followed by the removal of 900 μL supernatant, and the remaining liquid was used to gently resuspend the bacteria. The resuspended bacterial solution was used to spread the cells onto a LB solid medium plate (with 100 μg/mL ampicillin). The cells were incubated at 30°C, placed upright for approximately 10 min, then inverted for overnight culture. Single clones were selected as templates for colony PCR verification, and a gene fragment on the pKD46 plasmid was amplified using YZpkd46-F/R as upstream and downstream primers. A single colony that was correctly verified was cultured in LB liquid medium (with 100 μg/mL ampicillin) at 30°C and 200 r/min overnight. CGMCC 1.366/pKD46 competent cells were prepared by converting the knockout frame CK into CGMCC 1.366/pKD46 competent cells to obtain the recombinant strains CGMCC 1.366/pKD46-CK. Different single colonies growing on the above plate were selected, placed in 50 mL of corresponding resistant LB liquid medium, cultured at 30°C and 200 r/min, and knockout box Ck-F/R were used as the primers for PCR amplification verification. The verified strains (according to agarose gel electrophoresis) were selected and sent for DNA sequencing. Correctly verified clones were directly used for the next experiment. The temperature-sensitive plasmid pCP20 was transformed into the strain CGMCC 1.366/pKD46-CK to produce FLP recombinase (Xiao et al., 2022), which removed the kanamycin resistance gene. Finally, positive clones were screened by colony PCR. The homology of the fusion fragment was confirmed by DNA sequencing, and recombinant E. coli CGMCC 1.366-A was obtained.

panD and aspC were fused to cohesin and dockerin domains to drive their sequential co-immobilization on self-assembly systems. The inducible promoter Ptrc, aspC, cohA, panD and docA were cloned into pET28a (+) plasmid by selecting restriction enzyme sites EcoR I and Not I. DocA is an intracellular mutant that has undergone activity verification. Li et al. (2022b) have confirmed that DocA can bind and assemble within cells, and improve product synthesis. The target fragment was cloned into a pET28a (+) plasmid. Two plasmids were constructed based on the different assembly modes of the key elements of the cellulosome. Plasmids pET28a (+)-aspC-cohA-panD-docA and pET28a (+)-panD-cohA-aspC-docA were synthesized by Shanghai Sangon Biotech. The unassembled recombinant plasmid pET28a (+)-aspC-panD-docA was constructed by inverse PCR to verify the assembly effect of cellulosomes using the recombinant plasmid pET28a (+)-aspC-cohA-panD-docA as a template, the target gene pET28a (+)-aspC-panD-docA was amplified by reverse PCR. The PCR amplification conditions involved predenaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 1 min, the process of denaturation, annealing and extension was cycled 30 times, followed by a final extension at 72°C for 5 min. The products obtained by reverse PCR amplification were digested with Dpn I, and the target bands were recovered using a DNA gel recovery kit (Vazyme, Nanjing, China) to obtain the recombinant plasmid pET28a (+)-aspC-panD-docA. The construction design of these three plasmids is shown in Supplementary Figure S1. Finally, recombinant plasmids were electrotransformed into E. coli CGMCC 1.366-A competent cells to construct engineered E. coli CGMCC 1.366-B with vector pET28a (+)-aspC-cohA-panD-docA, E. coli CGMCC 1.366-C with vector pET28a (+)-panD-cohA-aspC-docA, and E. coli CGMCC 1.366-D with vector pET28a (+)-aspC-panD-docA. Primers YZcohA-F/R and YZdohA-F/R were used for PCR amplification. Verification strip as shown in Supplementary Figure S2. Compare and observe the size and shape of the band presented on the DNA gel map through the band of the DNA marker used to judge whether the band is correct. The strains verified to be correct were directly subjected to the next experiment of 50% glycerol preservation.

Verification of induced expression of cohesin and dockerin: E. coli CGMCC 1.366-B, E. coli CGMCC 1.366-C, and E. coli CGMCC 1.366-D were inoculated in 50 mL liquid LB medium containing 50 μg/mL kanamycin and cultured at 37°C and 200 r/min in a constant temperature shaker until an OD₆₀₀ of 0.8. Isopropyl-β-D-1-thiogalactopyranoside [IPTG, Sangon Biotech (Shanghai, China)] was added to a final concentration of 0.2 mM. This bacterial solution was centrifuged at 37°C and 12,000 r/min for 20 min, and the cells were collected. The cells were washed in 8 mL 0.2 M phosphate buffer (pH = 7), trypsinized, and an ultrasonic disrupter (Ningkai Instrument, Nanjing, China) was used to disrupt the bacterial solution for 20 min each time, wherein each test comprises 6 times of stimulation of 4 s and rest time of 6 s. Protein expression was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE, BIO-RAD (United States)]. Protein binding was determined by native polyacrylamide gel electrophoresis [Native-PAGE, BIO-RAD (United States)].

The verified strains were inoculated into a medium containing kanamycin (50 μg/mL) and ampicillin (100 μg/mL) in 50 mL liquid LB medium and grown at 37°C for 8–12 h. One milliliter of this culture was used to inoculate 100 mL sterilized liquid fermentation medium and grown until OD₆₀₀ = 0.8, followed by induction at 28°C using 0.2 mM IPTG. Fermentation was carried out for 60 h, and samples were collected to measure β-alanine production by high-performance liquid chromatography (Pawellek et al., 2020); the glucose and L-lysine content was determined using a biosensor analyzer (Yanhe Biotechnology, Jinan, China). We also optimized the induction temperature. E. coli CGMCC 1.366-C was induced at 22°C, 25°C, 28°C, 31°C, and 34°C at 200 r/min for 20 h to explore the optimal induction temperature for the fermentation of the engineered E. coli strains.

Fermentation was performed in a 5 L fermenter by inoculating the activated strain in 200 mL liquid LB culture medium containing kanamycin (50 μg/mL) for 8–12 h, then 10% of the culture was used to inoculate 5 L sterilized liquid fermentation medium. It was confirmed that the previous inducer, 0.2 mM IPTG, was replaced by 5 g/L lactose. Lactose inducer was added when OD₆₀₀ = 3 and fermented at 28°C for 20 h and then fermented at 37°C for 40 h. Samples were periodically taken to detect β-alanine.

β-alanine was detected by high performance liquid chromatography (HPLC). First, the sample was treated with 2,4-dinitrofluorobenzene (DNFB) for pre-column derivatization (Qu et al., 2021). One hundred microliters of the sample was taken, followed by the sequential addition of 100 μL 0.5 mol/L sodium bicarbonate solution (pH = 9.0) and 100 μL 1% 2,4-dinitrofluorobenzonitrile. The solution was mixed well, and placed in a water bath protected from the light at 60°C for 1 h, cooled at room temperature, followed by the addition of 700 μL 5 mM phosphate buffer (pH = 7.0), and centrifuged at 12,000 r/min for 5 min. The supernatant was passed through a 0.22 μm microporous membrane, and transferred to a liquid chromatography sample vial for later use. The HPLC instrument was high performance liquid chromatography-1 (Shimadzu LC-20 A, Japan). The chromatographic column was a Shim-pack GIST C18, and the sample was separated using an isocratic method using 1:1 mobile phase A:B, where mobile phase A and B is sodium acetate (2.5 g/L sodium acetate, 1.5 mL acetic acid, and ultrapure water to 1 L) and pure methanol, respectively. The flow rate was 1 mL/min, the column temperature was 40°C, the injection volume was 10 μL, the ultraviolet wavelength was 360 nm, and the detection time was 20 min.

CGMCC 1.366 is an E. coli engineered bacterium that has a yield of L-lysine, and aspartate kinase plays a decisive role in the synthetic metabolic pathway of L-lysine. Aspartate kinase was knocked out to block the synthesis of L-lysine to improve the accumulation of β-alanine. The aspartate kinase gene knockout strain (E. coli CGMCC 1.366-A) was obtained after homologous recombination, positive selection, and PCR detection. The upper and lower homology arms (lysC1 and lysC2) and the FRT site fragment were (FRT-Kan-FRT) amplified by PCR. The agarose gel electropherograms of the fragments are shown in Supplementary Figure S3A–C. Lanes 1–4 in Supplementary Figure S3A are agarose gel electropherograms of the PCR-amplified homologous arm fragment on lysC, which is 500 bp in length. Lanes 1–4 in Supplementary Figure S3B are agarose gel electropherograms of the PCR-amplified homologous arm fragment under lysC, which is 500 bp in length. Lanes 1–4 in Supplementary Figure S3C are agarose gel electropherograms of the PCR-amplified FRT-Kan-FRT fragment, which is 1,324 bp in length. Lanes 1–6 in Supplementary Figure S3D are agarose gel electropherograms of the double-digested plasmid by selecting restriction enzyme sites Nde I and Hind III, which is 5,306 bp in length. Lanes 1–6 in Supplementary Figure S3E are agarose gel electropherograms of the knockout fusion fragment, which is 2,324 bp in length. Lanes 1–6 in Supplementary Figure S3F are agarose gel electropherograms for a gene fragment validation of pKD46 plasmid, which is 775 bp in length. Lanes 1–6 in Supplementary Figure S3G are agarose gel electropherograms of gene fragments on pCP20 plasmid, which is 660 bp in length. M represents the DNA marker. E. coli CGMCC 1.366-A was fermented for 60 h, with a β-alanine yield of 0.529 mg/L. Under the condition of 250 mg/L L-lysine initial concentration, the L-lysine production was 1,510 mg/L after 60 h fermentation. E. coli CGMCC 1.366 secreted 1,260 mg/L L-lysine, and the remaining L-lysine content of E. coli CGMCC 1.366-A strain was 30 mg/L, the result showed that E. coli CGMCC 1.366-A could not synthesize L-lysine. This indicates that the lysC gene of E. coli CGMCC 1.366 was successfully knocked out.

In the metabolic pathway for β-alanine production, L-aspartate aminotransferase expression for the production of L-aspartate and co-expression with L-aspartate-α-decarboxylase for the production of the L-aspartate derivative β-alanine, the optimal enzyme for increasing β-alanine production, were selected as the L-aspartate-α-decarboxylase of B. subtilis. Three recombinant vectors are constructed and key enzymes in the metabolic pathway of the synthetic β-alanine are assembled by system of key elements of cellulosomes (cohesin and dockerin). This method can realize the co-expression of multiple enzymes, shorten the transfer time between enzyme molecules, improve the catalytic efficiency of the enzymes, and further increase the product yield (Figure 2). To analyze the enzymatic properties of docA and cohA binding to panD and aspC genes, the expression plasmid pET28a (+) was used. Based on SDS-PAGE gel electrophoresis data, E. coli CGMCC 1.366-B successfully expressed cohA binding aspC and docA binding panD. The band of the protein is clear and obvious, the molecular weight of CohA-AspC protein and DocA-PanD protein was approximately 60.3 kDa and 21.4 kDa, respectively. E. coli CGMCC 1.366-C successfully expressed cohA binding panD and docA binding aspC. The molecular weight of CohA-PanD protein and DocA-AspC protein was approximately 30.5 kDa and 51.0 kDa, respectively. The molecular weight of aspC protein and DocA-PanD protein in E. coli CGMCC 1.366-D was approximately 43.6 kDa and 21.4 kDa, respectively, based on SDS-PAGE protein gel electrophoresis Figure 3. The molecular weight of CohA protein and DocA protein was approximately 16.7 kDa and 6.7 kDa, respectively (Supplementary Figure S4). In Native-PAGE protein gel electrophoresis (Supplementary Figures S5, S6), the bands in the red box on lanes 3-4 are larger than the bands on lanes 1-2, indicating that AspC and PanD were assembled in E. coli CGMCC 1.366-B and E. coli CGMCC 1.366-C.

Recombinant E. coli CGMCC 1.366-A was transformed with the construction plasmid pET28a (+)-aspC-cohA-panD-docA containing cellulosome assembly elements to construct recombinant E. coli CGMCC 1.366-B. The constructed plasmid pET28a (+)-panD-cohA-aspC-docA was transferred into recombinant E. coli CGMCC 1.366-A to construct recombinant E. coli CGMCC 1.366-C. The unassembled construction plasmid pET28a (+)-aspC-panD-docA was transferred into recombinant E. coli CGMCC 1.366-A to construct recombinant E. coli CGMCC 1.366-D. Recombinant E. coli CGMCC 1.366-B, E. coli CGMCC 1.366-C, and control E. coli CGMCC 1.366, CGMCC 1.366-A, and CGMCC 1.366-D were fermented in shake flasks. Glucose was used as the raw material in 100 mL of fermentation broth, then IPTG (0.2 mM) was added when the recombinant strain was cultured to OD₆₀₀ = 0.8. Fermentation was carried out for 60 h, and sampling was carried out at 12 h. The OD₆₀₀ detected by the spectrophotometer (Yuanxi Instrument, Shanghai, China) is shown in Figure 4. The residual L-lysine content and the detected glucose content obtained after the detection by the biosensor analyzer are shown in Figure 5. The content of β-alanine produced after HPLC (Shimadzu LC-20A, Japan) detection is shown in Figure 6. We found that the cell density of the five strains increased rapidly in the first 12 h. E. coli CGMCC 1.366-A, CGMCC 1.366-B, and CGMCC 1.366-C grew slowly compared with the wild type strain E. coli CGMCC 1.366 OD₆₀₀ = 3.685. Although the unassembled strain (CGMCC 1.366-D) grew faster and consumed more glucose. Whereas the synthetic content of β-alanine was almost the same as E. coli CGMCC 1.366 and CGMCC 1.366-A, which could hardly produce β-alanine and the content was low, which was 0.7103 mg/L. The residual L-lysine content produced by the knockout E. coli CGMCC 1.366-A and the recombinant CGMCC 1.366-B and CGMCC 1.366-C was almost 0 mg/L after 60 h of fermentation at the initial concentration of 250 mg/L L-lysine, indicating that the three strains no longer synthesized L-lysine. The β-alanine yield of recombinant E. coli CGMCC 1.366-B and CGMCC 1.366-C was significantly improved and the β-alanine content reached 7.439 mg/L and 25.87 mg/L, respectively after 60 h compared with the control strain that hardly produces β-alanine. The β-alanine content was 0.7103 mg/L compared with control E. coli CGMCC 1.366-D which was not assembled. The β-alanine yield of recombinant E. coli CGMCC 1.366-B and CGMCC 1.366-C were 10.47 times and 36.42 times than those of the unassembled control E. coli CGMCC 1.366-D. In the experiment of optimizing induction temperature, E. coli CGMCC 1.366-C was induced by IPTG at 22°C, 25°C, 28°C, 31°C, and 34°C, respectively. The engineered E. coli strain produced the best β-alanine content under induction at 28°C (Supplementary Figure S7). The production performance of E. coli CGMCC 1.366-C with high β-alanine content in fed-batch fermentation was studied. E. coli CGMCC 1.366-C was induced at a final concentration of 0.2 mM IPTG and 5 g/L lactose to verify the effect of the inducer on β-alanine synthesis in a fermentation experiment. The content of β-alanine was higher under lactose induction than that under IPTG induction (Supplementary Figure S8). With the increase of fermentation time, the initial concentration of glucose of 20.0 g/L was rapidly consumed to 5 g/L after 12 h. The fermentation time after feeding was between 12 h and 36 h and feeding every 6 h, the substrate was exhausted. The results showed that the substrate consumption of E coli CGMCC 1.366-C was fast in 5 L fermentor. The β-alanine content was 755.465 mg/L after 72 h of fermentation (Figure 7). The HPLC chromatogram of β-alanine synthesis by fermentation of E coli CGMCC 1.366-C, with the peak time of 5.976 min (Supplementary Figure S9). This indicates that the assembly system of key elements of cellulosomes promotes β-alanine synthesis.