Results and analysis

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1.Construction, testing of heterologous 3-HB production strains and screening of the thioesterase

Under tetracycline induction, the expression of Thioesterase TesB in Z.mobilis resulted in the highest yield of 3-HB (425.67±2.89 mg/L). Consequently, Thioesterase TesB was selected for subsequent strain transformation.

2.Replace the inducible promoter of 3-HB strain with a strong promoter and replace the high-intensity RBS

(1)By substituting the strong promoter, the production of 3-HB was reduced to 232.67±2.51 mg/L.
(2)Substituting RBS10K resulted in a decrease in 3-HB production to 405.333±28.989 mg/L (Figure 1).
Therefore, the utilization of inducible promoters and RBS10 was continued for subsequent strain modification.

Figure 1 the production of ethanol and 3-HB in strong promoter expression strain NP-PgT1、High-strength RBS expression strain NP-PtT1’

3.Construction and evaluation of multi-copy strains for the 3-HB pathway

The production of 3-HB was increased to 533.00±4.36 mg/L by amplifying the gene related to the 3-HB pathway.

4.Construction and testing of cofactor enhanced strains

The overexpression of the cofactor-related gene (zwf) resulted in a significant increase in 3-HB production, reaching 685.67±10.69 mg/L.

5.The 3-HB strain lacks penicillin-binding proteins (PBPs) that promote the secretion of 3-HB

The production of 3-HB increased to 723.33±9.71 mg/L following the elimination of penicillin-binding protein in the 3-HB strain (Figure 2).

Figure2 the production of ethanol and 3-HB in PBPs deficient strains NPΔ0959-PtZT1、NPΔ1089-PtZT1

6.Introducing exogenous ethanol utilization to increase the supply of acetyl-coA

The implementation of the exogenous ethanol utilization pathway (EUP) resulted in a significant enhancement in 3-HB production, with the yield reaching 1114.33±26.50 mg/L.

7.Optimization of culture medium enhances 3-HB production

The yield of 3-HB was enhanced to 1323±22.61 mg/L under a carbon-to-nitrogen ratio of 50/5 (Figure 3).

Figure 3 the production of ethanol and 3-HB in NPTΔ1-PtZT1 EUP in different C/N ratio

8.Construction and testing of effective strategy combination strains

The highest 3-HB yield achieved in this study was 1525±72.16 mg/L, which resulted from the effective implementation of strategic approaches and optimal medium conditions (Figure 4).

Figure 4 the production of ethanol and 3-HB in strain NPTΔ3-PtZT1EUP(a) in different C/N ratio

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Results and analysis

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Analysis of strain of hypoglycemic and hypohypertensive peptides

1.The production of GLP-1 peptides by the BL21 (DE3) strain

The constructed strain was verified by PCR method (Figure 1).

Figure 1

The constructed strain was verified through Sanger sequencing.

Figure 2

The sequencing results indicate that the constructed strain is correct (Figure 2).

Results of protein expression

Figure 3

Due to the excessive disorder in the gel map, where proteins of different sizes were present in almost every interval (Figure 3), we conducted a purification process for it.

Figure 4

By using imidazole solutions with different concentration gradients for elution, no impurities were observed when eluted with a 50mM imidazole concentration, the protein size is approximately 90kDa (Figure 4).

2.The roduction of the 4GLP-1-5LV therapeutic peptides by the Escherichia coli Nissle 1917 (DE3) strain

(1)The constructed strain was verified by PCR method (Figure 5).

Figure 5

(2)The constructed strain was verified through Sanger sequencing (Fiure 6,7).

Fiure 6 primer: PT7-SEQ-F

Figure 7 primer: T7-ter

The sequencing results indicate that the constructed strain is correct.

Results of protein expression

Figure 8

Figure 9

Figure 10

The target protein has a size of 24.97 kDa, in addition to the TrxA tag of 11.8 kDa, resulting in a total size of 36.77 kDa. The SDS-PAGE gel shows a protein band approximately at 45 kDa, and there is no significant difference between the control group before and after induction (Figure 8,9,10).

3.The production of 4GLP-1-5LV peptides by the ZMNP strain

(1)The constructed strain was verified by PCR method (Figure 11).

Figure 11

(2)The constructed strain was verified through Sanger sequencing(Figure 12)

Figure 12

(3)The sequencing results indicate that the constructed strain is correct.

Results of protein expression(Figure 13, 14).

Figuer 13 empty vector

Figuer 14 pTZ32a/pTZ28a-4GLP-1-5LV

4.The production of 4GLP-1-5LV peptides by the ZM4 strain

(1)The constructed strain was verified by PCR method (Figure 15).

Figure 15

(2)The constructed strain was verified through Sanger sequencing (Figure 16).

Figure 16 primer: PT7-SEQ-F/T7-ter

(3)Results of protein expression(figure 17, 18)

Figure 17 empty vector

Figure 18 pTZ32a/pTZ28a-4GLP-1-5LV

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