Part:BBa_K1668002

frr (from Streptomyces avermitilis, increasing avermectin production)

The part frr is the coding sequence of ribosome recycling factor (RRF) in Streptomyces avermitilis. It was found to promote cell growth and stimulate the production of avermectins, one kind of pesticide.
This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp [1] as its promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 165
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 337
  

Characterization

BACKGROUND

Fig.1 the 3D structure of ribosome recycling factor (Uniprot #:Q82JY0)

frr is the gene encoding ribosome recycling factor (RRF), which is responsible for the release of ribosomes from messenger RNA at the termination of protein biosynthesis.

Function

frr gene encodes the ribosome recycling factor (RRF), which is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. RRF may increase the efficiency of translation by recycling ribosomes from one round of translation to another. Avermectin yield was increased significantly by 3- to 3.7-fold in transformants 31267(pFRR-1139) and 31267(pFRRerm-1139), compared with that in the wild-type strains and both of the transformants contained multiple frr copies.The avermectin productivity of each culture was quantitatively measured by HPLC analysis.

Principle

Research indicated that frr overexpression promoted cell growth as well as the expression of ave genes (including pathway-specific positive regulatory gene aveR for avermectin biosynthesis and ave structural genes), leading in turn to avermectin overproduction. Different from S-adenosylmethionine synthetase gene (metK), frr gene revealed a ‘‘copy number effect’’. That is to say, multiple copies of frr had a greater promoting effect on avermectin production than a single copy does. However, the detailed mechanism of frr enhancing antibiotic production remains to be clarified.

Limitation

Compared with the wild-type strain, the effect of frr on avermectin production in engineered strains 76-02-e and GB-165 was less obvious, probably because most of the negative stimulatory factors are downregulated and most of the positive factors are upregulated, resulting in relatively limited potential for further improvement of avermectin yield.

Protein

The 3D structure of ribosome recycling factor is as above (Fig.1). This factor is responsible for the release of ribosomes from messenger RNA at the termination of protein biosynthesis. and may increase the efficiency of translation by recycling ribosomes from one round of translation to another.

RESULTS

Gel electrophoretic analysis

Fig.2 Gel electrophoretic analyses of PCR products (A) and selected examples of cloned products of seamless assembly reaction (B)

In Fig.2, (A) 5-μl samples of the PCR products for frr, (B) 5-μl samples of the bacteria solution PCR products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. The DNA size standards was the DL1,000 DNA Marker (M; Takara, Cat#3428A) . Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. PCR products, positive clones and negative clones are indicated.

DNA sequencing

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 384bp part shows 100% agreement with the desired sequence.

REFERENCE

Li, L., et al. (2010). "Overexpression of ribosome recycling factor causes increased production of avermectin in Streptomyces avermitilis strains." J Ind Microbiol Biotechnol 37(7): 673-679.
http://www.uniprot.org/uniprot/Q82JY0