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410 18 Methyltransferases in Biocatalysis

l-Met 7 and SAM 1, respectively. A similar increase in metabolic flux toward SAM 1
was achieved by recombinant expression of a chimeric gene for methylenetetrahy-
drofolate reductase in Saccharomyces cerevisiae [80], which increases availability of
5
the co-substrate of Met synthase, N -methyltetrahydrofolate (Figure 18.4).
Although yeasts have been proven to be excellent producers of SAM 1,coupling
of the cofactor metabolism to methyl transfer reactions has not been established yet.
As the vacuole contains the predominant part of cellular SAM, organelle-specific
targeting of the recombinant MTs, insufficient activity under acidic conditions,
and low accessibility of substrates seem to be major limitations. Prokaryotic hosts
might be much more suitable in this regard, but knowledge on engineering of
bacterial SAM metabolism is still limited. Similar to yeast, overproduction of SAM
synthase appears to increase the intracellular concentration of SAM in E. coli fed
with l-Met 7. Thus, in the biotransformation of quercetin 17 to rhamnetin 18 by
E. coli cells producing recombinant O-methyltransferase (OMT), a twofold increase
in methylation capacity was observed upon coexpression of SAM synthase [81].
Accordingly, further metabolic optimization is required to obtain tailored (living)
whole-cell biocatalysts with improved SAM 1 availability in the future.

18.2.5
Cascade Applications

In contrast to many hydrolytic enzymes (such as lipases or proteases), only a
limited number of methylating enzymes have been applied in synthetic chemistry.
Although MTs are valuable tools for chemo- and regiospecific modification of
natural products, dependence on the cofactor SAM limits in vitro applicability (see
Chapter 18.2.4). Hence, conversions involving MTs are often performed by addition
of the corresponding substrates to suspensions of microorganisms or plant cells
producing the enzyme. Advantageously, addition of SAM 1 is not required in these
live whole-cell biotransformations because the cofactor is provided by the host’s
endogenous metabolism. In combination with low costs for biocatalyst preparation
and environmental friendliness, those conversions proved to be beneficial for the
production of many valuable secondary metabolites such as phenylpropanoids and
other phenolics, or alkaloids (Table 18.2).
Phenylpropanoids are ubiquitous plant metabolites, which include flavonoids
and stilbenes. Both groups of phenylpropanoids share a related biosynthesis
comprising the generation of non-methylated intermediates and their subsequent
conversion into a huge variety of derivatives by hydroxylation, methylation, and
prenylation reactions. In the last years, the progress in synthetic biology has led to
the integration of those cascade reactions to valuable phenylpropanoids – especially
flavonoids – in microbial hosts. For example, rhamnetin 18 was produced from
quercetin 17 by biotransformation with transgenic E. coli strain harboring an OMT
enzyme specific for the 7-hydroxyl group and SAM synthase to assist in the supply
of the cofactor SAM 1 [81] (Table 18.2). Also, two-step enzyme reactions including
hydroxylation were developed. Upon introduction of flavonol hydroxylase activity
into E. coli, naringenin 19 was oxidized to the corresponding flavonol or flavone.