Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONThe generation of novel compounds can also be achieved by using secondarymetabolite biosynthetic genes cloned into plasmid vectors and heterologouslyexpressed in different actinobacteria or other microorganisms, which have a relaxedsubstrate specificity or possessing similar structures sharing parts of their biosyntheticpathways (Kealey et al., 1998; Salas & Mendez, 1998). Factors influencingheterologous production of secondary metabolites include: (i) correct posttranslationmodification ;(ii) broad range of substrates and supply of substrates coordinatelyregulated with SM biosynthesis when required; (iii) intracellular factors such aschaperones that ensure correct folding and assembly of SMBG; (iv) transmembranetransporter proteins such as the ATP binding cassette transporters which are requiredfor the export of secondary metabolites; (v) and self-resistance mechanism/s to inhibitthe effect of the SM on the heterologous host (Pfeifer & Khosla, 2001). The benefitsof expressing genes in a heterologous host are that easier genetic manipulation withregard to enhancement of commercial production and recombination of genes forrelated pathways to produce new natural products. Chary et al. (2000) demonstratedthat by using two strong heterologous promoters from S. griseus transferred into N.lactamdurans lead to the overexpression of the rate limiting lat gene and subsequentlyincreased the yield of the production of cephamycin C. A limitation of heterologousexpression is that gene clusters larger than 40 kb cannot be accommodated intocosmid cloning vectors and 100 kb for bacterial artificial chromosomes (BAC)vectors (Sosio et al., 2000b).Combinatorial biosynthesis is defined as the production of hybrid secondarymetabolites or analogues resulting from novel combinations of genes, achieved by theintroduction of genes from one organism into another host organism (Hutchinson,1997; Tang and McDaniel, 2001). The requirements for the production of hybridsecondary metabolites are that firstly, the availability of two or more known or newlydiscovered microorganisms that produce secondary metabolites whose biosynthesishas a common feature. The second requirement is that the biosynthetic pathway mustbe fully characterised, and thirdly a gene-enzyme connection must be established(Huchinson, 1999). Combinatorial biosynthesis can be approached in two ways togenerate a diverse range of new compounds with new activities. The first of theseincludes cloning of single or multiple genes encoding structural modificationreactions, such as those encoding methyltransferases and reductases that modify basic_____________________________________________________________________50