Part:BBa_K4808001

ilvA

ilvA gene encodes the threonine dehydratase which takes out water molecules and ammonium molecules from the threonine. Threonine dehydratase is an enzyme that can turn threonine into our target product, a-KB.

Characterization

In order to produce a-KB, we sought to express the ilvA gene within the AIS strains. The ilvA gene encodes the enzyme Threonine Dehydrase, which is responsible for the removal of water and ammonium molecules from threonine (as depicted in Figure 1A). We intended to use the IPTG-induced pTac promoter system to regulate the expression of the ilvA gene. However, since AIS series strains of E. coli are relatively underexplored. There was uncertainty regarding the functionality of this system in our context.

To ascertain whether the IPTG induction system operates effectively in AIS strains, we conducted a validation experiment using GFP as a signaling protein. This allowed us to assess the efficiency of the IPTG inducer and determine the optimal induction concentration. The results, illustrated in Figure 1B, confirmed that the AIS strain responded positively to the IPTG induction, with concentrations ranging from 0.1 mM to 0.2 mM exhibiting the strong inducing effect.In subsequent experiments, we used 0.3mM iPTG to induce gene expression, which is the most commonly used concentration in the laboratory.

With the IPTG induction system established, we proceeded to construct the PW1-ilvA plasmid for a-KB production. This process began by obtaining the ilvA gene fragment from the E. coli genome through PCR. Subsequently, we integrated this fragment with the PW1 vector using the Golden Gate recombination method. The successful construction of the PW1-ilvA plasmid was verified through colony PCR results (see Figure 1C) and gene sequencing outcomes (as shown in Figure 1D).

Figure1: (A) ilvA function and IPTG induced-pTac promotor system.(B)test the induction sysytem by different iPTG concentrations in AIS strains.(C)colony PCR to determine the construction of PW1-ilvA. (D)verified the construction result through sequencing.

We transformed the PW1-ilvA plasmid into strains AIS-0, AIS-1, AIS-2, and AIS-3, each with the aim of producing a-KB. Regrettably, our efforts to insert pw1-ilvA into AIS-3 were unsuccessful. We attribute this outcome to the simultaneous knockout of both the rthA and ilvBN genes, along with the overexpression of the ilvA gene, which appeared to hinder cell growth. We speculate that this inhibition may be due to the cytotoxic effects of a-KB.

For the successfully transformed strains, we conducted HPLC methods to confirm the successful production of a-KB. We acquired a-KB standards from the market, by comparing the peak diagram of the standard a-KB with the supernatant of the AIS fermentation broth, we confirmed the production of a-Kb.Then, we made a standard curve with a-kb concentration of 0-20g/L in order to calculate the production of a-kb in AIS series(figure 4B). Observing the bar charts below, we achieved the highest a-KB yield in the AIS-2 strain, about 1.8g/L.(figure 1C).

Figure 2: (A) HPLC peak diagrams of standard a-KB and AIS supernatant sample. (B) the standard curve about the relationship between peak area and a-KB concentration. (C)the production of a-KB in AIS series with pw1-ilvA plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1315
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1315
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1315
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1315
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1315
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1168
  

References：

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Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948