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18.2 SAM-Dependent Methyltransferases 409

that is sufficiently reactive but stable in aqueous environment or in the presence of
the nucleophilic substrates of MTs is not (yet) available.
Alternative to chemical alkylation, SAM is also accessible by enzymatic adenosy-
lation of l-Met 7 with ATP [65]. This reaction uniquely accounts for SAM synthesis
in all organisms as a part of the so-called SAM cycle [66] (Figure 18.4). It is
catalyzed by SAM synthase and yields (S,S)-SAM 1 only. However, as most SAM
synthases are inhibited by their product SAM 1 under in vitro conditions [34b,
63a, 67], only low conversion rates are achieved. Although this drawback might be
partially eliminated by medium engineering [68], the development of an efficient
process for the biocatalytic synthesis of SAM will greatly benefit from new enzymes
that are insensitive to inhibition and have a high specific activity. Moreover, ATP
regeneration has to be taken into account.
As in vitro regeneration of SAM 1 is currently not feasible, MT reactions were often
performed with live whole-cell biocatalysts to avoid the need of cofactor supply
(i.e., microbial hosts expressing recombinant MTs, see next section). However,
a drawback of this strategy is the relatively low intracellular concentration of
endogenous SAM 1, which limits methylation capacity and, thus, leads to low
product yield. Even in the biosynthesis of natural products in microorganisms, such
as methylated antibiotics [69] and triterpenoids [70] or fatty acid methyl esters [71], or
in the biotransformation of phenolic compounds by plant cell cultures, for example,
of protocatechuic aldehyde to vanillin 28 [72], availability of SAM 1 is rate-limiting.
Exceptional to the facts mentioned above, yeasts are able to accumulate high
intracellular levels of SAM 1 [62]. Unlike in other microorganisms, SAM 1 is
removed from the cytosol by efficient secretion into the vacuole. As the cofactor
can be easily extracted from disrupted yeast cells, this propensity to build cofactor
storage provides the basis for fermentative production of SAM 1. Preparations of
such yeast-derived SAM 1 (e.g., the stable toluene sulfonate salt which is marketed
under the trade name SAMe) are sold as a dietary supplement and for therapeutic
applications [73]. As recently reviewed by Chu et al. [74], the yield in SAM 1 has been
greatly improved in the last decades by screening for suitable strains. Outstanding
productivity was reported from a strain of Saccharomyces sake isolated by Shiozaki
and coworkers. In a medium supplemented with l-Met, the yeast produced up to
10.8 g l −1 of SAM 1 [75]. Improved production in nonmodified yeasts was achieved
by optimization of culture conditions and led to yields up to 13.2 g l −1 [76]. Addition
of choline to the growth medium also enhances SAM 1 production [77]. During
fermentation, choline seems to be converted into its oxidation product glycine
betaine, which supports SAM 1 biosynthesis by acting as a methyl donor in the
conversion of l-homocysteine 8 to l-Met 7 (Figure 18.4).
As various enzymes of the SAM cycle and of accompanying reactions (Figure 18.4)
are tightly regulated with respect to gene expression [66], accumulation of SAM
1 in yeast also proved to be an excellent target for metabolic engineering. Cofac-
tor production was significantly increased in Pichia pastoris by overexpression of
SAM synthase [78] or by influencing the SAM cycle indirectly via knockdown of
cystathionine-β-synthase [79]. This enzyme channels l-homocysteine 8 into the
adjacent transsulfuration pathway (Figure 18.4) and competes with recovery of