Chapter 13-Synthesis of commercial products by recombinant microbes Restriction enzymes Small molecules Antibiotic genes Biopolymers

Restriction enzymes are great products for recombinant microbes (E Restriction enzymes are great products for recombinant microbes (E. coli) $350 million in annual RE sales in 2007 Some microbes are difficult or expensive to grow in culture Strategy: clone the gene for the RE from a given microbe and express it in E. coli (along with the corresponding modification [methylase] gene for protection of the E. coli DNA) E. coli is simple to grow

Figure 13.1 Restriction enzyme and the corresponding methylase are usually on a single operon. The restriction enzyme is secreted to the periplasmic space and the methylase remains in the cytoplasm.

Figure 13.2 Method for cloning and selecting the gene for the restriction enzyme PstI

Figure 13.3 Method for cloning and selecting the gene for the Dde I restriction enzyme and the Dde I methylase

Small molecules are also great products for recombinant microbes (often E. coli) Indigo (Fig. 13.10) Ascorbic acid (Vitamin C) Amino acids (e.g., Glutamic acid or Glu for production of the flavor enhancer MSG) Antibiotics, novel antibiotics and polyketide antibiotics Note that in all of these cases, one needs to clone the genes encoding the enzymes making these metabolites in order to create or alter a biochemical pathway

Cloning amino acid biosynthetic genes Table 13.1 Cloning amino acid biosynthetic genes

Cloning antibiotic biosynthetic genes Table 13.3 Cloning antibiotic biosynthetic genes

Biopolymers are also great products for recombinant microbes Xanthan gum production in Xanthomonas compestris (genetically engineered to grow on whey, a lactose-rich byproduct of cheese production) Melanins Animal adhesive proteins (from the blue mussel) Rubber (from the rubber plant Hevea brasiliensis) Biodegradable plastics (polyhydroxyalkanoates) Note that in all of these cases, one needs to clone the genes encoding enzymes in order to create or alter a biochemical pathway

Figure 13.31 Structure of xanthan gum (glucose backbone with Man-GluA-Man trisaccharide sidechains)

Engineering of the E. col lacZ (encoding b-galactosidase) and lacY (encoding lactose permease) genes for constitutive expression in Xanthomonas campestris. Figure 13.32

Figure 13.36 The enzymes responsible for the production of this biodegradable plastic were cloned from Alcaligenes eutrophus and transferred to E. coli to make even more of this biodegradable plastic.

Summary I do not expect you to memorize all of the genetic engineering details provided in the numerous examples presented in this chapter; however, I do want you to be familiar with the various strategies that are employed in these examples. Specifically, note the common strategy that a gene for an enzyme is transferred into a microbe which alters the metabolic products of that microbe (i.e., metabolic engineering).