Limitations in Metabolic Engineering Due to Technology
As discussed earlier in this chapter, several steps are involved in metabolic engineering. We discussed the possibilities and achievements, but limitation are experienced at each step, which will be discussed briefly in this section.

Limitations of cloning in industrial strains
In many industrial strains, cloning is difficult due to non-availability of (i) suitable vectors and (ii) methods for introduction of exogenous DNA. Even after successful transfer, stable propagation of cloned genes is problematic as observed in Bacillus subtilis. This is due to rearrangement, deletion and cleavage of cloned sequences and the plasmid vector carrying it.

Measurements of concentrations of intermediate products also becomes necessary in some cases to find out the possible rate-determining reactions. Once these rate-determining reactions are known, genetic transfer of stress response motifs can be tried. Under these conditions, the use of organisms that can function over wide range of temperature and pH would help in the industrial production of a metabolite.