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414 18 Methyltransferases in Biocatalysis

Subsequently, regiospecific methyl transfer yielded 3-O-methylkaempferol 20 [82]
or genkwanin 21 [84] as the main products. Further pathway engineering, that
is, integration of the genes for p-coumaric acid-CoA ligase, chalcone synthase,
and chalcone isomerase from the flavonoid biosynthetic pathway, led to modified
cells which even synthesized methylated flavonoids such as genkwanin 21 [83] or
7-O-methylaromadendrin 22, a compound with anti-inflammatory and anticancer
activity [85], from p-coumaric acid 23. Similarly, an artificial biosynthesis for
stilbene production was constructed into E. coli by Katsuyama and coworkers
[86]. In a multistep reaction (consisting of an aromatic amino acid-ammonia
lyase, p-coumaric acid-CoA ligase, and stilbene synthase), the strain converted
phenylalanine or tyrosine into pinosylvin 24 and resveratrol 25, respectively. Methyl
transfer to these stilbenes, which are important dietary antioxidants, was catalyzed
by a sequential OMT enzyme from rice and yielded the mono- and dimethyl ethers.
Recently, even transgenic tobacco plants capable of synthesizing pinosylvin ethers
were generated [87].
Especially, those phenolics bearing a vanilloid (4-hydroxy-3-methyoxyphenyl)
motif can be potent flavoring substances. A number of studies focus on the bio-
catalytic manufacturing of vanillin 26, the world’s most important flavor originally
isolated from fermented Vanilla orchid seed pods. For example, an engineered path-
way that involves human catechol-OMT was described in 1998 by Li and Frost [88].
Starting from 3-dehydroshikimic acid, an intermediate in the biosynthesis of aro-
matic amino acids, the pathway affords the production of vanillic acid from glucose
in E. coli. However, reduction of vanillic acid to the final product vanillin 26 necessi-
tated its isolation from the culture medium and application of purified aryl aldehyde
dehydrogenase from Neurospora crassa. In integrated processes, which were per-
formed with transgenic yeasts, the biocatalytic cascade includes this reduction
step [89]. Highest yields were achieved upon coexpression of a glycosyltransferase
(Table 18.2) which transforms vanillin 26 into its less toxic glucoside [90].
Similar to many phenylpropanoid (phenolic) compounds, the biological activity
of alkaloids originates from specific methylation patterns. Thus, the biosynthesis of
alkaloids relies on the assistance of both OMTs and N-methyltransferases (NMTs).
Reaction sequences to alkaloids are often highly complex and require the collabo-
ration of different plant organs. Therefore, alkaloid production in microorganisms
proved to be challenging. A decade ago, synthetic biology of alkaloids was restricted
to single-step modifications such as methyl transfer to the imino group of tetrahy-
droisochinoline 27 [91]. Since then, progress in the identification of genes as well
as the development of new tools in molecular biology has led to the assembly
of whole pathways in microorganisms or crop plants in a synthetic biology type
approach. Functional expression of key enzymes from morphinane biosynthesis
enabled the accumulation of the opium alkaloids salutaridine 28 and reticulin
29 (Figure 18.5) in S. cerevisiae [94]. With the objective to increase resistance to
insects, caffeine biosynthesis was transferred into tobacco [92]. Recent efforts to
engineer alkaloid metabolism also focus on the generation of transgenic plants
with tailored MT activity. For example, overexpression of putrescine-NMT, which