Part:BBa_K3832010

pykA (pyruvate kinase II)

This part encodes pyruvate kinase II (E.C. 2.7.1.40) in E.coli DH5a and K-12 substrain.

Reaction:

ATP + pyruvate = ADP + phosphoenolpyruvate

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 953
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 953
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 606
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 953
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 953
  
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution by Tianyu Sun

Summary

Compared with the old part of pyruvate kinase (PK, BBa_K3832010), we have increased the application range of pyruvate kinase. Probiotics can now produce pyruvate kinase to generate short-chain fatty acids (SCFAs) and help in the treatment of depression. We have also added a new part, pGEX-4T-1-PK (BBa_K5190003), and supported this with extensive test data.

The experimental test data are focused on the following aspects:

• We constructed the recombinant plasmid pGEX-4T-1-PK and used heat shock transformation into E. coli DH5α to preserve the plasmid.
• We performed protein expression in E. coli BL21 and identified the successful expression of the target protein using SDS-PAGE.
• We conducted in vitro enzyme activity assays for pyruvate kinase (PK) to more intuitively demonstrate the changes in enzyme activity in our engineered strains.
• Finally, we transformed the recombinant plasmids (pGEX-4T-1-PK) into E. coli ECN and used high-performance liquid chromatography (HPLC) to detect the content of propionate.

Engineering Principle

Propionic acid salt levels in the intestine are believed to be associated with various diseases, including depression. Attempts to regulate gut microbiota to increase propionic acid salt levels to improve conditions are ongoing. Once successful, such research will benefit hundreds of millions of people globally.

Short-chain fatty acids (SCFAs) refer to fatty acids containing 5 carbon atoms or fewer, such as formic acid, acetic acid, propionic acid, butyric acid, and valeric acid, which are typically present in the intestine in salt form¹. Current research focuses mainly on acetate, propionate, and butyrate salts. Due to the functional overlap and interconversion among these salts, supplementation of a single component can also effectively supplement short-chain fatty acid salts².

We have analyzed the metabolic pathways and identified the key pyruvate kinase (PK) related to SCFAs (Figure 1). We aim to enhance the production of short-chain fatty acids in the strain by overexpressing the key pyruvate kinase (PK) in order to further achieve the purpose of aiding the treatment of depression.

Figure 1. Metabolic Engineering Diagram of SCFAs (Red Indicates Overexpression)

Construction Design

The pGEX (BBa_K5024006) plasmid was provided by our institution's strain repository, and the target gene PK (BBa_K3832010) was synthesized by a biotech company. Using the pGEX plasmid as a template, the PK fragment was homologously recombined with a linearized plasmid to construct pGEX-4T-1-PK (BBa_K5190003) (Figure 2).

Figure 2. The plasmid map of pGEX-4T-1-PK

Characterization/Measurement

1. Protein Expression

We performed protein expression in E. coli BL21 and E. coli ECN. SDS-PAGE was used to identify whether the target protein was successfully expressed. The successful expression of the target protein is demonstrated by the protein bands observed in SDS-PAGE experiments.

2. Functional Test

Ⅰ: In Vitro Enzyme Activity Assays for Pyruvate Kinase

We conducted in vitro enzyme activity assays for pyruvate kinase (PK) to intuitively demonstrate the changes in enzyme activity of our engineered strains. The enzyme activity of PK was measured using a standard PK enzyme assay kit, and the results show a significant enhancement in PK activity in our modified strains.

Ⅱ: HPLC Detection of Propionate Content

We transformed the recombinant plasmid pGEX-4T-1-PK into E. coli ECN and performed fermentation. Using high-performance liquid chromatography (HPLC), we monitored the propionate content over time in both the control and experimental groups. The data show an increase in propionate content in the engineered strain, further demonstrating the successful production of propionate by the engineered strains.

References

¹ Silva, Y.P., Bernardi, A. & Frozza, R.L. The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication. Front Endocrinol (Lausanne) 11, 25 (2020).
² Fan, L., et al. Gut microbiota bridges dietary nutrients and host immunity. Sci China Life Sci 66, 2466-2514 (2023).
³ Tomar, A., Eiteman, M.A. & Altman, E. The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli. Appl Microbiol Biotechnol 62, 76-82 (2003).