Part:BBa_K2571003

FucO L-1,2-propanediol oxidoreductase

L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase.

FucO is the gene that codes for L-1,2-propanediol oxidoreductase which is a NADH-linked, homodimer enzyme having the role of acting on furfural which is a toxic inhibitor of microbial fermentations causing cell wall and membrane damage, DNA breaks down and reduced enzymatic activities (Zheng, 2013; Liu, Ma & Song, 2009).

The enzyme catalyzes L-lactaldehyde and L-1,2- propanediol while dissimilating fucose in which acetaldehyde, ethylene glycerol, L-lactaldehyde and some more substances are used as substrates. Despite these, it takes an important role in furan reduction to its alcohol derivative (Wang et al. , 2011).

Source

Gene : FucO Protein : L-1,2-Propanediol Oxidoreductase Organism : E.coli K12 (MG 1665)

Our circuit design for FucO gene

Our circuit consists of prefix, a strong promoter (J23100), RBS (B0034), FucO as protein coding region, double terminator (B0015) and suffix. This part enables our E. coli KO11 strain to convert toxic furfural into furfuryl alcohol. Our construct is inserted into pSB1C3 and delivered to the Registry.

Circuit design of BBa_K2571003. Our construct includes a strong promoter, RBS, Fuco and double terminator.

In order to make our gene compatible with RFC 10, 25 and 1000, we reconstructed the nucleotides to get rid of the restriction sites while protecting the amino acid sequence. We looked through the codon bias property of E. coli and made the nucleotide changes accordingly.

FucO has NADH-dependent furan reductase activity. When furfural is present in the field, the metabolism of furfural by NADPH-dependent oxidoreductases go active in order to reduce it to its less toxic alcohol derivative-furfuryl alcohol (Zheng, 2013; Wang et al., 2013; Allen et al., 2010).

Effect of FucO overexpression in LY180 (Wang et al. , 2011). The Cell Mass was observed in furfural containing medium. The FucO gene expressing L-1,2-propanediol oxidoreductase reduce the effect of furfural. The specific death rate of normal bacteria is observed to be bigger than the specific death rate of bacteria with FucO gene. Thus FucO shows to increase the tolerance of bacteria and lifespan.

In this metabolism, the expression of oxidoreductases that are NADPH-dependent, such as YqhD, are shown to inhibit the growth and fermentation in E. coli by competing for biosynthesis with NADPH (Zheng, 2013).

[[File:METU_HS_Ankara Furfural pathway.png|600px|thumb|center|The overexpression of FucO and YqhD and relationships with furfural resistance traits, metabolism, and reducing cofactors (Wang et al. , 2013).

Because the native conversion of NADH to NADPH in E. coli is insufficient to revitalize the NADPH pool during fermentation, the actions shouldn’t be interfering with NADPH metabolism (Wang et al. , 2011). Thus, the overexpression of plasmid-based NADH-dependent propanediol oxidoreductase (FucO) gene reduces furfural to ultimately improve furfural resistance without detrimentally affecting the biosynthesis of NADPH (Wang et al., 2011).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]