Biocatalyst genetic engineering

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

In this section, we will consider the methodologies used for genetic engineering of biocatalysts for desulfurization and the biocatalysts developed so far via various technologies. The application of genomic techniques as reported in patent literature associated with BDS is described first. 

It should be noted that Diversa was involved in a BDS study from 2003 to 2006 in collaboration with PetroStar, Inc. They acquired many of the strains developed by EBC and investigated the potential of desulfurizing diesel using these strains and conducted further genetic engineering and host strain manipulation to develop better biocatalysts. 

Conversions of primary feedstocks by fermentations (such as glucose to ethanol) are not included in this book. However, fermentations are usually required to produce the enzymes or cells in the first place, and therefore chapter 5 includes a review of this type of fermentation. Chapter 5 also covers the other aspects of biocatalyst production, except immobilization and protein and genetic engineering, which are treated in chapter 6 and 7, respectively. 

Genetic engineering techniques to improve penicillin amidase yields during fermentation are now employed thereby reducing biocatalyst process costs. 

Genetic engineering to improve biocatalyst/process. HFCS, (S)-2-chloropropanoic acid ... 

Physiological optimization of enzyme synthesis by variation of the culture parameters is usually required to enhance the catalytic activity of whole-cell biocatalysts to such a level that it can be apphed in a biocatalytic process. In addition, physiological conditions can influence the selectivity of the reaction, since enzymes with opposite selectivities can be differentially expressed. In some cases, genetic engineering is required to obtain biocatalysts with a desired selectivity that does not consume the product of choice (see 5.3.5). Alternatively, one may choose to isolate the desired activity from the culture in order to use the biocatalyst in an enzyme reactor. 

Whether a biocatalyst consists either of an isolate from a natural environment, of a classically improved strain or of a strain improved by various types of genetic engineering, the biocatalyst that is obtained after a screening effort will eventually be applied in a process. In order to obtain sufficient amounts of active biocatalyst, the cells have to be produced by fermentation. 

The use of a recombinant strains as biocatalysts has the advantage that once the fermentation conditions for the host microorganisms have been found, the conditions to be used for the recombinant strain are mostly close to these. Furthermore, the need for induction can be removed, undesired side reactions can be avoided and enzymes from pathogenic strains can safely be produced in recombinants of GRAS organisms. Therefore, biocatalysts that are amenable to genetic engineering are frequently preferred over those that are not. 

With the advances in genetic engineering technology, directed evolution is no longer rocket science. It is a proven technology which opens a fast and relatively inexpensive pathway for developing new biocatalysts [97]. Successful synthetic... 

Despite this limitation, in vitro biosynthesis using purified biocatalysts promises to be a simple and inexpensive means to access a wealth of complex natural product structures under clean and controlled conditions. The versatility of this technology can be significantly enhanced by the application of genetic engineering to create novel proteins. 

---