Progress in the Chemistry of Cytochalasans
97
4.3 Future Prospects
The most important breakthrough to be anticipated in future chytochalasan biosynthesis work would be to achieve pyrrolinone formation and DA cycloaddition by
heterologous expression. Since the main problems are found from the unexpected
side reactions occurring in the common heterologous hosts, feasible solutions may
include either finding enzymes in these heterologous hosts that catalyze the side
reactions and delete these genes or finding new heterologous expression host strains
that do not cause unwanted modifications. These problems may be resolved in the
near future and an expression system found that can provide cytochalasin core structures. This will help to characterize more cryptic BGCs than have been discovered to
date and facilitate the engineering of biosynthetic pathways to give additional new
compounds.
In addition, due to the presence of unsaturation on ring C of the cytochalasans,
further structural diversification could be targeted. For example, chaetoglobosins
generally have a double bond at C-21/C-22 conjugated with adjacent carbonyl groups.
At this site, multiple heteromolecular adducts are formed (e.g., in 334–336 and 340–
342 in Fig. 14). It can be speculated that the C-21/C-22 double bond can be attacked
conditionally by nucleophilic reagents such as cysteine, or can readily participate in
[3 + 2] or [4 + 2] cycloadditions with unsaturated small molecules (e.g., epicoccine
and aureonitol). Although enzymatic catalysis cannot be ruled out at this stage,
these addition reactions are likely to be spontaneous, and other hybrid molecules
could be formed by feeding nucleophiles or unsaturated compounds suitable for
cycloadditions.
As many BGCs have been discovered, combinatorial biosynthesis will help
produce further cytochalasan derivatives. As mentioned earlier, the recombination
of cytochrome P450s in different BGCs has produced unnatural derivatives. Other
enzymes with novel activities, including CcsB that catalyzes iterative oxidations to
form carbonates, could participate in the recombination of modifying enzymes to
produce novel derivatives. In addition to the enzymes in the later stages, there are
also studies for engineering PKS-NRPS enzymes through domain swapping and
heterologous expression [252]. Although newly assembled products were obtained,
the cytochalasan backbone was not formed due to the heterologous expression problems mentioned above. Thus, if the side reaction issue in the heterologous hosts
could be solved, it will be of great interest to engineer the early stage biosynthesis
of cytochalasans.
The three-stage biosynthesis of cytochalasans demonstrates the considerable
ability of microorganisms to produce highly diversified metabolites. Studying
biosynthesis not only will reveal interesting metabolic reactions, but also will
inspire bioengineering and biomimetic synthesis work to obtain further interesting
cytochalasan derivatives.
97
4.3 Future Prospects
The most important breakthrough to be anticipated in future chytochalasan biosynthesis work would be to achieve pyrrolinone formation and DA cycloaddition by
heterologous expression. Since the main problems are found from the unexpected
side reactions occurring in the common heterologous hosts, feasible solutions may
include either finding enzymes in these heterologous hosts that catalyze the side
reactions and delete these genes or finding new heterologous expression host strains
that do not cause unwanted modifications. These problems may be resolved in the
near future and an expression system found that can provide cytochalasin core structures. This will help to characterize more cryptic BGCs than have been discovered to
date and facilitate the engineering of biosynthetic pathways to give additional new
compounds.
In addition, due to the presence of unsaturation on ring C of the cytochalasans,
further structural diversification could be targeted. For example, chaetoglobosins
generally have a double bond at C-21/C-22 conjugated with adjacent carbonyl groups.
At this site, multiple heteromolecular adducts are formed (e.g., in 334–336 and 340–
342 in Fig. 14). It can be speculated that the C-21/C-22 double bond can be attacked
conditionally by nucleophilic reagents such as cysteine, or can readily participate in
[3 + 2] or [4 + 2] cycloadditions with unsaturated small molecules (e.g., epicoccine
and aureonitol). Although enzymatic catalysis cannot be ruled out at this stage,
these addition reactions are likely to be spontaneous, and other hybrid molecules
could be formed by feeding nucleophiles or unsaturated compounds suitable for
cycloadditions.
As many BGCs have been discovered, combinatorial biosynthesis will help
produce further cytochalasan derivatives. As mentioned earlier, the recombination
of cytochrome P450s in different BGCs has produced unnatural derivatives. Other
enzymes with novel activities, including CcsB that catalyzes iterative oxidations to
form carbonates, could participate in the recombination of modifying enzymes to
produce novel derivatives. In addition to the enzymes in the later stages, there are
also studies for engineering PKS-NRPS enzymes through domain swapping and
heterologous expression [252]. Although newly assembled products were obtained,
the cytochalasan backbone was not formed due to the heterologous expression problems mentioned above. Thus, if the side reaction issue in the heterologous hosts
could be solved, it will be of great interest to engineer the early stage biosynthesis
of cytochalasans.
The three-stage biosynthesis of cytochalasans demonstrates the considerable
ability of microorganisms to produce highly diversified metabolites. Studying
biosynthesis not only will reveal interesting metabolic reactions, but also will
inspire bioengineering and biomimetic synthesis work to obtain further interesting
cytochalasan derivatives.