Revision as of 12:31, 11 October 2018

ProR-PhaR (The phasin autoregulation system with native promoter)

This is a composite part designed to investigate the role of the phaR autoregulation system on cell growth and PHA production. PhaR is the core member of the phasin autoregulation system, which contribute to the PHA operon and PHA production. It is known to repress the expression of the phasin through binding to the phasin promoter and auto-regulate its own expression. Otherwise, phaR can also bind to the PHB granules. With the coexpression of the PHA operon in recombinant E.coli, this part has verified its novel function in direct improving the PHA production, even without the phasin.

Usage and Biology

According to previous research (Pötter et al, 2002), phaR binds at two sites upstream of phasin. One site is the transcriptional initiation site plus the -10 region. The other site is a region just upstream of the -35 region of the σ70 promoter of phaP. Gene of phaR was also recognised to bind to 86 bp upstream of the start codon in the open reading frame (ORF) of phaR. In addition, PhaR was detected binding on the surface of PHA granules (Pötter et al, 2002; Yamada et al, 2013). Based on the binding behaviours and following influence, it was confirmed that phaR regulated not only phaP but also itself precisely (Pötter et al, 2002).

During the later stages of PHA accumulation, PhaR leaves the PHA granules as the surface of PHA granules is mostly covered with phasin. The replaced PhaR returns to bind to the phaP promoter and represses transcription of phasin again. PhaR has been reported to autoregulate the expression of itself by binding on its promoter(Yamada et al, 2013).This autoregulation system guarantees the efficient expression of phasin as well as curtailing excessive expression of the PHA biosynthetic pathway, decreasing the metabolic burden imposed on cells.

Experiments and Results

Recombinant E.coli harbouring phaCAB operon + phaR autoregulation system with and without phasin were constructed to investigated the influence of phaR autoregulation system on PHB production and cell growth rate. Recombinant E.coli harbouring psb1C 3 + P. Furthermore, HlyA depending secretion was reported to function in recombinant E.coli for PHB secretion. The protein fusion of phasin and HlyA was expressed for PHB secretion via Type I secretion system. The influence of HlyA depending secretion on PHB was also investigated.

The effect of PhaR on cell growth

Figure 1. The growth curve of recombinant E.coli strains.

Comparing the PHA production of E. coli expressing new construct

Figure 2. . Nile red plates culture to confirm PHB production (48 hours). In each picture of a, b, c, d, e, there were 4 Nile red plates. The left plates on the first rows were strains harbouring phaCAB operon and constructs. The right plate on the first row were strains containing the construct only. The second row were strains harbouring phaCAB operon only as well as pSB1C3and pSB3T5 backbones.phaCAB operon strain showed strong signal of fluorescence because of PHB produced and the backbone strain did not due to no PHB produced.

PHA Extraction and Melting temperature measurement

Table 1. The yield of PHB production

Figure 3. Data for dry weight of intracellular and secreted PHB.

Figure 4. Melting temperature of PHB produced by recombinant E.coli strains.

Sequence and Features