Part:BBa_K4849032

dapA - aspartokinase

Diaminopimelate biosynthesis A: dapA

 

Description

The enzyme aspartokinase has been studied in Escherchia coli (E. coli) as a crucial enzyme catalysing the phosphorylation of the amino acid aspartate, being an initial step occurring prior to the biosynthesis of lysine [1]. This enzyme could therefore be involved in the biosynthesis of lysine in several bacteria, such as in E. coli and Cyanobacteria. A study by Nærdal et al. (2011) looked into the engineering of genes involved with the aspartate pathway for an overproduction of lysine by Bacillus methanolicus (B. methanolicus) from methanol, identifying key genes involved in lysine production within this bacterium. The gene diaminopimelate biosynthesis A, dapA, encoding the enzyme dihydrodipicolinate synthase (DapA), was specifically a central gene involved in increasing lysine production which could be involved in the first stages of lysine biosynthesis (see fig. 3) [2]. Essentially, this gene is associated as the rate-limiting step in the biosynthesis of lysine [3]. Expression from the dapA promoter is regulated by intracellular diaminopimelic acid [4].

 

The catalysed reaction consists of the condensation of pyruvate and (S)-aspartate β-semialdehyde. This is thought to be the rate-limiting step in lysine biosynthesis after aspartate kinase III. The product of the reaction catalysed by DapA was identified as (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate (HTPA). The reaction proceeds via a ping-pong bi-bi mechanism; pyruvate initially binds to the enzyme via a Schiff base to the ε-amino group of the active site Lys161 residue. This is followed by addition of L-aspartate semialdehyde and transimination leading to cyclization and dissociation of HTPA. The kinetic mechanism was refined using initial velocity and dead-end inhibition studies at both high and low pH, confirming the ping-pong reaction mechanism of the enzyme.[5]

Figure 1 biosynthesis pathway of dapA

 

Lysine use in Pollen Substitute

Declining floral pollen availability due to environmental changes, particularly rising temperatures, poses a threat to crucial pollinators like honeybees. To address this, overexpression of the dapA gene in Cyanobacteria is explored as a solution to enhance lysine levels, aligning them with those found in natural pollen. This approach aims to provide an alternative and sustainable source of essential amino acids, crucial for the growth and health of pollinators.

 

Figure 2: The concentrations of essential amino acids in various diets for pollinators, including pollen. Ultra Bee refers to a commercial plant-based pollen substitute

 

 

Characterisation

 

Ninhydrin

 

Ninhydrin finds application in numerous bioanalytical methods, especially in the analysis of amino acids. It engages with the α-amino group of primary amino acids, resulting in the formation of 'Ruhemann's purple.' The chromophore generated is consistent across all primary amino acids, with the colour intensity varying based on the quantity and chemical characteristics of the amino groups under examination. The most favourable pH for the entire reaction is 5.5, and Ruhemann's purple exhibits its spectral peak at 570 nm. By normaling OD₅₇₀ ninhydrin results to OD600’s of overnight cultures of test samples a quantitative comparison can be made between samples for relative levels of amino acids present. We used this test as a quantitative indication of increased amino acid expression. Whilst the test is insufficient to distinguish between individual amino acid levels, the increase of amino acids can be associated with overexpression of dapA by comparison to a negative control with a baseline level of dapA expression.

 

Figure 8: Ninhydrin test on dapA overexpressed strain (Table 1). Shows 2% amino acid standard solution (2% AA), Wild type (WT) and dapA level 1 overexpressed strain (dapA). dapA shows a 1.48x increase compared to WT. Error bars are standard deviations.  p=4.73E-5.

Table 1: Data from ninhydrin test. Average OD570 of samples normalised to sample overnight culture OD600 for comparative results.

 

3 different dapA colonies were picked from the level 1 transformation plate following cPCR validation and from each colony 3 overnight cultures were set up making a total of 9 samples for ninhydrin analysis(Figure 8). Triplicates of WT overnight cultures were set up. Protocol followed as documented below and samples were heated for 20 minutes at 90°C using a water bath. Table 1 summarises the normalised average for the samples.
 

There is a clear improvement in amino acid levels, and by extrapolation lysine, with a 1.48x increase compared to a wild type control. Whilst this increase cannot be directly translated to a proportional increase in lysine levels, there is strong evidence that overexpressing dapA leads to increased lysine levels. Further metabolomic quantification is required for an individual amino acid breakdown.

 

 

 

Figure 9: Comparison of WT and dapA overexpressed strain ninhydrin results following 20 minutes at 90°C. Clear colour differences can be seen between the two samples, notably a darker purple hue for dapA, indicating the presence of Ruhemann's purple in greater quantities as a result of increased lysine levels.

 

 

 

Further Metabolomics

Samples were sent off for mass spectrometry analysis however due to delays in receiving results they were unable to be received in sufficient time. Mass-spectrometry (MS) analysis is a reliable technique used for the identification and quantification of amino acids, including lysine. MS allows for the analysis of mass-to-charge ratio (m/z) of ions generated from the sample. In the context of lysine quantitative analysis, the sample containing these amino acids is usually first subjected to protein hydrolysis or acid hydrolysis to break down proteins or peptides into individual amino acids. Next, the resulting amino acid mixture is derivatised to enhance ionisation efficiency in the mass spectrometer. Once the sample is prepared, it’s introduced into the mass spectrometer, where the amino acids are ionised, and m/z ratios are measured. The mass spectrometer can provide accurate mass measurements, enabling the identification of specific amino acids based on their unique m/z values. Quantification of lysine could be achieved by comparing the intensities of their respective signals to those of known standards or internal standards added to the sample, with isotope-labelled internal standards often being used for accurate quantification [7].

 

Expected changes in the cell
Given how the gene dapA may already be residing within Cyanobacteria, an increase in gene expression would hypothetically enhance processes that dapA are already performing within the lysine biosynthesis pathways of the bacterium – as seen below in fig. 10. We can expect that the increased use of ASM for lysine biosynthesis may lead to reduced methionine and threonine levels due to reduced substrate levels. To confirm this metabolomic m/z data would be required for quantification of individual amino acid levels.  

 

 

Figure 10: Proposed increased activities of relevant genes within the BPL of Cyanobacteria for this project. Stages show lysine biosynthesis in E. coli, adapted from [2], annotations by author.
 

References

1. Toney, M.D. (2014) ‘Aspartate aminotransferase: an old dog teaches new tricks’, Archives of biochemistry and biophysics, 54: 119–127
2. Nærdal, I. et al. (2011) ‘Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus’, Applied and environmental microbiology 17: 6020–6026
3. Laber, B., F. X. Gomis-Rüth, M. J. Romão, and R. Huber. 1992. “Escherichia Coli Dihydrodipicolinate Synthase. Identification of the Active Site and Crystallization.” Biochemical Journal 288: 691–95
4. Acord, John, and Millicent Masters. 2004. “Expression from the Escherichia Coli dapA Promoter Is Regulated by Intracellular Levels of Diaminopimelic Acid.” FEMS Microbiology Letters 235: 131–37
5. Karsten WE. Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine. Biochemistry. 1997 Feb 18;36(7):1730-9. doi: 10.1021/bi962264x. PMID: 9048556.
6. Vasudevan, R. et al. (2019) ‘CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax’, Plant physiology 39–55
7. Verrastro, I. et al. (2015) ‘Mass spectrometry-based methods for identifying oxidized proteins in disease: advances and challenges’, Biomolecules, 2: 378–411

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 64
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]