Mutant screening

Figure 4. Photograph of a two ml deep well plate with MicroMap used for the mutant screening.

Coniferaldehyde (3.76) can undergo several fates, some of which can ultimately lead to the same end product. It can be reduced to coniferyl alcohol (3.79) by the enzyme cinnamyl alcohol dehydrogenase (CAD). Alternatively, the enzyme coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (C5H), also known by its less accurate name ferulic acid 5-hydroxylase (F5H Humphreys et al., 1999) can catalyze the hydroxylation of C5 to result in 5-hydroxyconiferyl aldehyde (3.77). C5H is also able to form 5-hydroxyconiferyl alcohol (3.80) from coniferyl alcohol (3.79). This enzyme was initially identified as F5H, after analysis of the Arabidopsis ferulic acid hydroxylase 1 (fahl) mutant, which was isolated in a mutant screen based on reduced levels of the UV-fluorescent sinapoyl esters (Section 13 Chappie et al., 1992). The FAH1 gene was cloned using a T-DNA tagged mutant allele (Meyer et al., 1996), which revealed that the... 

The elucidation of sinapoyl ester metabolism was aided by the availability of mutants. The sngl sinapoyl glucose accumulator 1) mutant of Arabidopsis had been identified based on a mutant screen for alterations in the composition of fluorescent compounds in the leaves. The screen was performed by thin layer chromatography and revealed that the leaves of the sngl mutant contained less sinapoylmalate and instead accumulated the precursor sinapoyl glucose (Lorenzen et al. 1996). 

Fig. 6. The optimal DNA mutation rate as determined from a model that incorporates one-body and two-body fitness contributions (similar to a spin glass). The genetic code is included in the model. The data are for a N = 50 protein. The fitness improvement is the maximum change in fitness averaged over 10,000 landscapes. To compare the relative location of the optima, the curves have been scaled such that the optima are at 1.0. (a) The optimum mutation rate for the uncoupled landscape as the number of mutants screened increases M= 1000 (O), 10,000 ( ), and 50,000 (A), (b) The optimal mutation rate as the landscape ruggedness increases. The number of coupling interactions is 75 (O), 25 ( ), and 0 (A). As the landscape ruggedness increases, the optimal mutation rate decreases. Reprinted from Voigt et ol. (2000a), with permission.

Fig. 7. The probability distribution P(c) of a positive mutation with c coupled interactions occurs as the sequence ascends the fitness landscape (generated using a spin-glasslike fitness function). The distribution is shown at two positions on the fitness landcape, a random sequence (O) and a highly optimized sequence (A). As the sequence is optimized, the probability that positive mutations will be made at uncoupled residues increases considerably. The mutation rate is an average of one amino acid (three nucleotides) per sequence and the number of mutants screened is 3000. Reprinted from Voigt et at. (2000b), with permission.

In the IVK-model, as K increases, the number of fitter neighbors decreases more quickly as the sequence becomes more optimized (Kauffman and Weinberger, 1989). Thus, in order to discover improved mutants, the number of mutants screened has to increase more rapidly on random landscapes as the sequence increases in fitness (Fig. 4). The rate of decrease for the number of uphill paths is greater for rugged landscapes due to the shortening of the walk length to local optima. This implies that a protein that is tolerant (a smoother landscape) can undergo more rounds of mutation and improvement. 

Fig. 14. Predicted correlation between the improvement in fitness achieved W and the average mutation rate (Npm) for different numbers of mutants screened for the D130N mutant of catalase I. The number of mutants screened (from top to bottom) is 3 X 105, 3 X 104, 3 X 103, 3 X 102, and 3 X 10. A clear optimum is observed for all the screening library sizes at around Npm = 1. Reprinted from Matsuura et al. (1998) with permission.

In avermectin Bl, there is a double bond between C-22 and C-23, whereas in avermectin B2 R OH. The product ratio of B2 to Bl is 1.6 1. Since the Bl analog is much more effective as an antiparasite, there is a desire to improve the B1 B2 ratio, which can be achieved by a dehydratase domain [88, 89]. Its directed evolution and mutant screening led to a mutant giving an excellent fermentation titer of Bl B2 = 1 0.07 [90, 91]. As a result of understanding the biosynthesis, pathway engineering, and directed evolution, a biologically superior new product, doramectin (Dectomax ), was evolved from a mixture of eight natural products. 

Random mutagenesis + in vivo recombination + site-directed mutants + screening Yeast [123]... 

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