Part:BBa_K2260000

phaCBA operon with polyhistidine tags

Overview

The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) (Hiroe et al., 2012). It utilizes acetyl-coA, which is a product of the glycolysis pathway (Hiroe et al., 2012). Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA (Hiroe et al., 2012).

In order to utilize acetic acid and undigested glucose present in fermented human feces, we decided to incorporate the phaCAB operon. However, literature has shown that the rearrangement of operon to phaCBA leads to higher amount of production of PHB (Hiroe et al., 2012). Thus, we obtained the operon from BBa_K1149051 and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.

PHB Weights from fermented "syn poo" supernatant

Our part was tested for production of PHB using the PHB synthesis using VFAs as feedstock protocol. There were 9 replicates for our construct in the "syn poo" supernatant and 3 replicates of the negative control (E. coli (BL21) transformed with pET29(b)+, no insert). The OD₆₀₀ of overnights (O/Ns) was measured before inoculating the media with "syn poo supernatant". PHB was extracted using the sodium hypochlorite extraction protocol. After resuspension of cells in 1x PBS for extraction the OD₆₀₀ was recorded to estimate the number of cells. The following table summarizes the data:

Table 1. Recorded OD₆₀₀ of O/Ns before inoculating the media containing "syn poo supernatant", OD₆₀₀ of cells after growing in media for ~24 hours, centrifuged, and resuspended in (1x) PBS for extraction. Initial weight of 50 mL falcon tubes and final weights of tube containing PHB was recorded in grams.

Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on left and phaCBA on right.

HPLC of PHB digested in sulfuric acid

Karr et al. showed that digestion of PHB in sulphuric acid leads to the production of crotonic acid (1983). The protocol for using this digestion method for HPLC is included in PHB digestion in sulphuric acid protocol. About 0.581 g of product was obtained and digested for 20 mins (Low) and 30 mins (High). The dilution factor used for these samples was 100. Lastly, a sample with a dilution factor of 32 was digested for 30 mins. Figure 2 shows the HPLC results obtained for the sample with a dilution factor of 32.

30 mins digestion, dilution factor 32

Figure 1. HPLC results from digestion of PHB in sulphuric acid for 30 mins.

A standard curve was generated to determine the conversion of PHB to crotonic acid by digesting PHB from Polyferm. The amount of crotonic acid from HPLC results was then used to calculate the concentration of PHB in the sample.

The HPLC results showed that crotonic acid and therefore PHB was present in all the three samples. The dilution factors were selected based on the predicted conversion of 80% from PHB to crotonic acid. The concentration of crotonic acid could not be determined for the dilution factor of a 100 as it was outside the standard curve range. The amount of crotonic acid detected in the sample with a dilution factor of 32 was 0.0282006 mM. The dilution factor of 32 was an arbitrary value selected because the amount of PHB in product was unknown.

Discussion of HPLC results

The HPLC results confirmed that the product obtained was PHB. Although we were successful at detecting PHB using HPLC, the obtained amount of crotonic acid may be lower than the actual amount produced as the method of quantification of PHB using HPLC requires further development. In particular, an optimal digestion time needs to be selected so that all of the PHB in the sample digests to crotonic acid without further degradation of crotonic acid. The PHB extraction method can also be optimized to minimize the amount of PHB lost during extraction.

References

Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T. 2012. Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl. Environ. Microbiol. 78:3177–3184. 10.1128/AEM.07715-11.

Karr DB, Waters JK, Emerich DW. Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection . Applied and Environmental Microbiology. 1983;46(6):1339-1344.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]