Revision as of 03:36, 13 December 2019

phaC1-A-B1 [P(3HB) synthesis]

Poly-3-hydroxybutyrate, P(3HB), a kind of PHAs is synthesized by three enzymes.

• The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
• The B1 gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)
• The C1 gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)

To synthesize P(3HB) by E.coli, we transformed E.coli JM109 with the constructed phaC1-A-B1 parts on pSB1C3 (BBa_K934001). E.coli JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB). As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.

Fig.1 synthesis mechanism of P(3HB)

The pathway and regulation of Poly[(R)-3-hydroxybutyrate] ,P(3HB) synthesis in Ralstonia eutropha H16 is shown in Fig1. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in phaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in phaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).

Fig.2 shows the difference between cells storing P(3HB) and those not storing P(3HB). The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control.

We cultured the colony in LB solution for 16hrs at 37℃, then we concentrated the solution and painted the letter by the solution on LB agar medium including 0.5μg/ml Nile red and 2% glucose at 37℃ for 36 hours. The cells with P(3HB) would be stained red by Nile red when observed under UV.

Fig2. Difference between cells storing P(3HB) and cells not storing P(3HB). Blue rectangle: with BBa_K934001 gene, P(3HB) accumulation. Green rectangle: with PlasI-gfp gene, no P(3HB) accumulation.

We successfully identified the products by BBa_K934001 as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry (GC/ MS). To confirm the products using GC/ MS, the products are methylated because 3HB is difficult to measure. Fig.3 shows the GC/ MS result of the products by BBa_K934001. The peaks of sample are same to those of standard control of methylated 3HB. This shows that E.coli synthesized P(3HB) correctly.

Fig3. Result of GC/MS

Optimization of the best culture condition to synthesize P(3HB)

To figure out best culture condition, we tried culturing E.coli JM109 in 10 different conditions for 48h. Each condition is shown in Fig.5. Composition of LB and TB medium is shown in Fig.6.

Fig5. 10 conditions

Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA.

Fig6. Composition of LB & TB

Fig7. Structure of Pantothenic acid

The culture result is shown in Fig.8.

Fig8. culture result

• “Dried cells (g/L)” is the amount of the cells in the medium after culturing.

• “Polymer content rate (%)” is the rate of the polymer in the dried cells.

• “Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing. This value is calculated by multiplying “Dried cells” and “Polymer content rate”.

The results showed that TB medium was much better than LB medium to synthesize P(3HB). In both LB and TB, in the 37°C culturing containing glucose and PA-Ca, E.coli synthesized the polymer in maximum content rate. However, the growth of E.coli in 37°C was worse than that in 30°C, therefore final polymer concentration in 37°C and 30°C didn’t make a significant difference. Even if there was no glucose, E.coli synthesized polymer (condition 9 & 10). We think that TB medium had glycerol and a lot of yeast extra, and then E.coli might have used them as carbon sources.

In addition, the comparison of condition 4 & 5 indicates PA-Ca was not used as carbon sources. LB medium didn’t contain many carbon sources, so E.coli synthesized little polymer. In this case, adding PA-Ca didn’t have big effect. On the other hand TB medium contains enough carbon sources, so we think that the rate-limiting step was the glycolytic pathway. In this case, polymer production would be increased by adding PA-Ca. (the comparison of condition 7 & 8 and 9&10)

We made P(3HB) sheets. To make the sheets, we cultured E.coli JM109 in erlenmeyer flasks at 37℃ for 72h.

Fig4. P(3HB) sheet

For more information, see Experience, or [http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#3. our work in Tokyo_Tech 2012 wiki].

Aanerobic conditions test

BBa_K934001 biobrick was introduced in E. coli BL-21 and was cultured under aerobic and anaerobic conditions to test the growth and synthesis rate. Both media were inoculated with pyruvate 50mM to simulate the real conditions under our bacteria would work. This quantity was used because is necessary to suppress the pyruvate dehydrogenase complex repressor (pdhR) ensuring the theoretical maximum expression rate of pyruvate dehydrogenase complex which allows the metabolism of pyruvate into acetyl-coA, the first substrate in PHB synthesis.

Figure 1: PHB production under aerobic and anaerobic conditions

Results

After 72 hours we collected 25mg of PHB from the media with oxygen and pyruvate, however, we collected 90mg of PHB under anaerobic conditions and pyruvate.

Conclusion

This biobrick would work fine in our theoretical conditions, producing PHB in anaerobic conditions taking advantage of the high pyruvate production from E. coli.

Sequence and Features