Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONengineering strategies used for modifying SMBG include; (1) gene transfer: (2) genereplacement; (3) chromosomal insertion; (4) Homologous recombination; (5) DNAShuffling and (6) silent gene induction.1.8.2 Metabolic Engineering of Secondary Metabolite ProducingActinobacteriaMetabolic engineering is defined as the directed improvement of product formation orcellular properties through modification of specific biochemical reactions orintroduction of new ones with the use of recombinant DNA technology(Stephanopoulos, 1999). Rational strategies have focused on the tightly regulated firstbiosynthetic steps which control the metabolic flux through SM biosyntheticpathways, major emphasis has been placed on directed genetic alteration of geneswhich has lead to the overproduction of a SM or alteration of a pathway to produce anew product that could either be a intermediate or a modified end product(Piepersberg, 1994; August et al., 1999). The strategies used in metabolic engineeringof secondary metabolites, (i) include supplying precursors to control rate of productsynthesis and amounts of different analogs; (ii) preferential specificity of certainstarter units of biosynthetic pathways; (iii) incorporation of synthetic intermediates;(iv) control of chain length and cyclization; (v) and induced post-modificationreactions (Chartrain et al., 2000). Table 18 outlines the metabolic engineeringstrategies employed for secondary metabolite production. Manipulation of generegulators or activators can also be used to improve the flux rates through secondarymetabolic pathways (Charter, 1990).In cases where the biosynthesis of a SM is completely defined biochemically andgenetically, flux balance models can be used to dictate the changes needed to shift themetabolic pathway utilization from producing biomass towards producing higheryields of the SM of interest (Neaimpoor & Mavituna, 2000; Varma & Palsson, 1994).Improvements of general cellular properties, such as the ability to withstand hypoxicfermentation conditions (Cedrone et al., 2000), as well as inducing constitutiveexpression of resistance genes preventing self-inhibition of the actinobacteria andenhancing secondary metabolite productivity (Crameri et al, 1986)._____________________________________________________________________49