Team:Tec Guadalajara/Molecular Biology

The pathway from glucose to IA is already known in Penicillium. It consists of two steps catalyzed by two enzymes.

  1. Oxidation of D-glucose to D-glucono-1,5-lactone by glucose oxidase (GOX)
  2. Oxidation of D-gluconolactone to D-isoascorbic acid by D-gluconolactone oxidase (GLO)
Isoascorbic acid pathway

D-isoascorbic acid biosynthetic pathway

Both genes were optimized for Pichia pastoris and synthesized. Our chassis was methylotrophic yeast Pichia pastoris GS115.

Gene optimization

  • No P. pastoris strains to overcome codon bias
  • Codon optimization strategies are usually based on Kazusa's codon usage database
  • We used the COOL web tool using Bai et al's (2011) codon table based on highly expressed genes

Vector design

Design of our shuttle vector for E. coli and P. pastoris

Design of our shuttle vector for E. coli and P. pastoris

  • Strong constitutive expression
  • Same strength as PAOX1
  • One-step cultivation procedure
  • Shortened protein production time (no induction)
  • No glucose, alcohol, or glycerol repression
  • Effective secretory expression
    • pre-region: nascent protein is directed to the endoplasmatic reticulum post-translationally
    • pro-region: protein is transferred to Golgi apparatus
  • Eases protein purification and qualitative analysis
  • Cis-acting hydrolase element
  • Expresses multiple proteins in a single ORF
  • Already used for co-expression in P. pastoris
  • Single transformation
  • Balanced coexpression
  • Overall cost is reduced

Advantages of biological reduction of GO

The production of isoascorbic acid by using P. pastoris allows us to:

  1. Reduce GO without compromising operator's health and environment
  2. Scale-up the process. However, it must be improved to be economically feasible
  3. Show proof of concept to move from a chemical to a biological process