Part:BBa_K5366044

AJC7/S125D/T181A/ I129T/ L140P

AJC7 four-point mutant

Construction

Primers for the S125D point mutation were designed, and PCR was conducted using pET-28a(+)-AJC7 as the template (Fig. 1). Following PCR, demethylation was performed using DpnI. A 5 µL aliquot of the digested product was analyzed via nucleic acid gel electrophoresis for verification. Upon confirming the correctness of the product, we proceeded with PCR product recovery to obtain the single-point mutant plasmid. The concentration of the single-site mutant plasmid was measured, and it was subsequently transformed into E. coli BL21 (DE3) and incubated in an inverted culture at 37°C for 14 hours. From the transformed colonies, single colonies were selected for colony PCR. After verifying the results through nucleic acid electrophoresis, the corresponding single colonies that exhibited the correct bands were transferred to LB (Kan) liquid medium for preservation. This marked the completion of the S125D single-site mutation step. Following the successful creation of the S125D single-point mutant plasmid, we proceeded to generate the S125D/T181A two-point mutant plasmid using the same single-point mutation protocol. Subsequently, the S125D/T181A plasmid was further mutated to obtain the S125D/T181A/I120T three-point mutant plasmid. Finally, this three-point mutant plasmid was mutated to produce the S125D/T181A/I129T/L140P four-point mutant plasmid, which was validated through nucleic acid electrophoresis after being transformed into E. coli BL21 (DE3) (Figure 2).

Fig.1 Mapping of mutant plasmids

Fig.2 Nucleic acid gel diagram of colony PCR (4-point mutation on the left and 5-point mutation on the right)

Product Analysis

The mutant and wild-type strains were activated, cultured for amplification, and subjected to a series of protein purification processes to extract the target proteins, following the method outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The reaction system was set with a final fructose concentration of 100 g/L and included 10 µL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, after which the products were analyzed using High-Performance Liquid Chromatography (HPLC) (Figure 3).

Result

In an effort to enhance substrate transformation capabilities, we conducted the S125D/T181A/I129T/L140P four-point mutation on AJC7. The results indicated that the product concentration of the S125D/T181A/I129T/L140P four-point mutant was higher than that of the wild-type strain. However, when assessing the optimal mutant variants, we observed a decrease in product concentration. These findings suggest that while the S125D/T181A/I129T/L140P four-point mutant may exhibit improved activity relative to the wild type, it ultimately does not prove to be an effective modification for optimizing substrate transformation.

Fig.30 The concentrations of tagatose in WT, S125D, S125D/T181A, S125D/T181A/I129T, S125D/T181A/I129T/L140PS125D/T181A/I129T/L140P/H342L after reacting with 100g/L fructose substrate for 5 h

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 501
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1003
  
• 1000
  COMPATIBLE WITH RFC[1000]