Molecular techniques for microbial biocatalysts
The molecular techniques that have revolutioned the area of microbial biocatalysts include the following: (i) Expression cloning; (ii) molecular screening; (iii) protein engineering and directed (or artificial) evolution. These will be briefly discussed.

Expression cloning.
Microbial biocatalysts in many cases are discovered and produced using expression analysis of a microbe followed by cloning of the positive DNA sequence. Following steps are sequentially involved: (i) fungi of interest is fermented (ii) biomass is used for extracting mRNA; (iii) cDNA library is constructed in E. coli, using S. cerevisiae ­E. coli shuttle binary vector; (iv) plasmid DNA is isolated from subpools of library; (v) plasmid DNA is transformed into S. cerevisiae (yeast); (vi) yeast transformants are replicated onto agar plates with enzyme substrate to allow detection of enzyme producting transformants;

Molecular screening.
Genes encoding specific enzymes can also be detected by designing degenerate PCR primers from conserved sequences followed by use of these primers for PCR amplification of genomic DNA. Fungal genes encoding cellulases belonging to family 45 (Fam45) have been detected in many fungi using this approach. Initially, based on activity based assays, these enzymes were detected in five fungi, including Humicola insolens and Fusarium oxysporum. When sequences of Fam45 cellulases from these five microbes were aligned, it was found that the sequences differed, but contained well defined conserved regions. These conserved regions were used for designing degenerate primers. When these primers were used for screening 200 diverse fungi, 56 were found to have Fam45 genes.