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 BUAP_Mexico 2019

In order to test the PHB synthesis rate in E. coli under anaerobic and aerobic conditions as well as the bacterial growth, E. coli BL-21 was transformed using the biobrick BBa_K934001 and was cultured in TB medium. Aerobic and anaerobic 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.

Each 24 hours we measured the bacterial growth of both media and after 72 hours the bacterial culture was centrifugate until get a pellet, which wasstored at -20°C during 72 hrs, after this time chloroform was added to get the PHB. The measure of PHB production showed a production of 25mg in media with oxygen and 90mg of PHB under anaerobic conditions.

**Figure 1:** PHB production under aerobic and anaerobic conditions

**Figure 2:** Bacterial growth under anaerobic and aerobic conditions

Conclusion

The result showed that PHB in E. coli works better under anaerobic contions even if the number of microorganism is less tha the aerobic conditions. We think this biobrick would work fine in our theoretical conditions where there are no oxygen and exist a high concentration of pytuvate, in our experiment we confirm this molecule enhances the PHB production because under high concentration it repress genes related to aerobic pathways, in this conditions the pyruvate obtained is metabolized in PHB.

For more information see our wiki.

Concordia_Shanghai 2024's Characterization

Background

Our project further increased the functionality of this part by showing the successful synthesis of P(3HB) can be broken down into the ketone body β-Hydroxybutyrate (BHB). Our work added significant opportunities for expansion on the uses of this part and opened these genes up a new area of research. This part was successfully used for production of P(3HB) in our system (BBa_K5165002), and an additional application of phaC1-A-B1 was demonstrated for BHB production when linked to TALFU_PhaZ (BBa_K5165000). Our project’s primary goal was to produce β-Hydroxybutyrate (BHB); our main construct produces PHB using the phaC1-A-B1 sequence, which is then extracellularly depolymerized by TALFU_PhaZ into BHB. BHB is a ketone ester, an alternative energy source that potentially enhances athletic, cognitive, and cardiovascular performance when taken as a supplement.

Results

The high presence of BHB in our experiments indicates that PHB was successfully produced using BBa_K934001 and subsequently degraded, thereby confirming the functionality of this part for PHB production and confirming the produced PHB’s ability to be converted to BHB. We first conducted an assay to determine a standard curve for the concentration of BHB. The BHB colorimetric assay kit was purchased from Grace Biotechnology and standard curve was determined following the kit protocol. E.coli BL21 cells were successfully transformed with our designed plasmids. Colonies of each cell type from selection plates were grown overnight in liquid broth + ampicillin. The BHB colorimetric assay was carried out according to kit manufacturer's protocol where cells were separated from the growth media by centrifugation (12000rpm) for 10 minutes. Supernatant was collected and kit reagents added to each experimental group as per protocol.

The absorbance level of cells containing plasmid 1 (BBa_K5165002), plasmid 2 (BBa_K5165003), and wild type cells. Verifies the production of PHB and degradation into BHB (high absorbance indicating high levels of BHB production) and the functionality of BBa_K934001.]]

The results indicate a high level of BHB in the extracellular fluid of cells containing the plasmid with BBa_K5165002, indicating endogenously produced AHL was upregulating the expression of PHAZ_TALFU in comparison to trace amounts found the extracellular space of cells transformed with plasmid 2. Using the standard curve of BHB concentration (y = 0.4651x - 00221) we determined the average concentration of BHB in the extracellular space of cells transformed with plasmid 1 to be 1.347 umol/mL compared to just 0.138 umol/mL in the extracellular space of cells transformed with plasmid 2.

This means that the PHB has been produced in relatively high amounts using this part and has been secreted and degraded into BHB through other components in our system.

The lack of extracellular BHB in Plasmid 2 is unrelated to expression of this part or the functionality of these PHB producing enzymes; manual induction of this other system (BBa_K5165003) using AHL was likely unsuccessful due to errors in AHL preparation.

Figure 2. Qualitative data from β-Hydroxybutyrate colorimetric assay.Tube 1 contains kit reagents + a known quantity of standard solution (2 umol/mL). Tubes 2, 3, & 4 contains kit reagents + supernatant of cell environment for BL21 cells transformed with plasmid 1, plasmid 2, and wild type (untransformed cells) respectively. Expression of the system containing BBa_K934001 resulted in high amounts of extracellular BHB, meaning PHB was successfully produced intracellularly, then secreted and degraded by other components of our system.]]

This demonstrates that an additional application of these PHB synthesizing enzymes is for BHB production when linked to TALFU_PhaZ (BBa_K5165000) and Phasin-HlyA (BBa_K2260002), such as in BBa_K5165002.

Sequence and Features

Assembly Compatibility: