## 3 Technology Developments for Production of Carotenoids
### Protein Engineering for Improvement of Caroenoid Production Enzymes  
Pathway engineering for efficient production of desired chemicals is often challenged by limitations associated with the pathway enzymes themselves, such as low turnover numbers and promiscuities generating unwanted by-products [121]. Protein engineering provides a powerful solution to improve specific activity and substrate specificity of enzymes, and even to create new activity. Methods of protein engineering include directed evolution and computer-assisted rational design (Figure 5D) [122; 123]. Directed evolution is an iterative process that imitates Darwinian evolution in the laboratory to select or screen a desired phenotype from mutagenesis. Typically, error-prone polymerase chain reaction (PCR) is used to generate mutant libraries, and DNA shuffling is carried out to recombine existing mutations. It can be performed in a blind manner with limited information on target enzymes, such as structures and reaction mechanisms, but it relies on an effective screening strategy. It is practical for the evolution of carotenogenic enzymes due to the innate traits of carotenoid pigments. Six mutants ((H96L, R203W, A205V, A208V, F213L and A215T) have been isolated to improve the catalytic activity of \(\beta\)-carotene ketolase from _Sphingomonas sp_. [124]. Three mutations (L175M, M99V, and M99I) of ketolase from _Paracoccus sp_. result in the improvement if its specificity of to synthesize astaxanthin [125]. _Staphylococcus aureus_ dehydrogenase (C30) synthase has evolved to synthesize lycopene by mutation F26L or F26S [126]. DNA shuffling of phytoene desaturases from _P. agglomerans_ and _P.ananatis_ results in the isolation of a variant favoring the production of fully conjugated tetradehydrolycopene [127]. Rational design of proteins is based on the _in silico_ simulation and the prediction using _a priori_ enzyme information, which greatly liberates biologists from onerous screening task. This strategy requires adequate information to predict specific targeted amino acid mutations, which can confer desired enzyme traits [128]. Unfortunately, the limited information on carotenogenic enzymes leads to few achievements using such a method.  
As aforementioned, carotenoids are derived from the central isoprenoid pathway, which is also employed to synthesize several essential and secondary metabolites in nature. The carotenoid-based-colorimetric screening has been developed for evolution of other isoprenoid pathway enzymes. Mutations of GGPP synthase are hypothesized to affect the binding efficiency of the magnesium ions needed for substrate anchoring and improve its catalysis. An error-prone PCR library of _Tsuga canadensis_ GPPS has been screened using the lycopene synthesis pathway as a colorimetric reporter. The GPPS variant (S239C and G295D) is created to increase levopimaraadiene production with a 1.7-fold increase over the wild type in _E. coli_[129]. Augmentation of one pathway can tamper with other pathways, which utilize the same substrate in one organism. Based on this fact, mutagenesis libraries of terpene synthases have been screened by depigmentation of colonies due to the competition between terpene synthases and carotenoid synthases for isoprenyl diphosphates, since the weakened carotenoid color intensity indicates an improvement of terpene synthase activity [130].