Abstract
The 3-(3-hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA is an essential enzyme involved in the biosynthesis of HAAs in
Pseudomonas
and
Burkholderia
species. RhlA modulates the aliphatic chain length in rhamnolipids, conferring distinct physicochemical properties to these biosurfactants exhibiting promising industrial and pharmaceutical value. A detailed molecular understanding of substrate specificity and catalytic performance in RhlA could offer protein engineering tools to develop designer variants involved in the synthesis of novel rhamnolipid mixtures for tailored eco-friendly products. However, current directed evolution progress remains limited due to the absence of high-throughput screening methodologies and lack of an experimentally resolved RhlA structure. In the present work, we used comparative modeling and chimeric-based approaches to perform a comprehensive semi-rational mutagenesis of RhlA from
Pseudomonas aeruginosa
. Our extensive RhlA mutational variants and chimeric hybrids between the
Pseudomonas
and
Burkholderia
homologs illustrate selective modulation of rhamnolipid alkyl chain length in both
Pseudomonas aeruginosa
and
Burkholderia glumae
. Our results also demonstrate the implication of a putative cap-domain motif that covers the catalytic site of the enzyme and provides substrate specificity to RhlA. This semi-rational mutant-based survey reveals promising ‘hot-spots’ for the modulation of RL congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between the
Pseudomonas
and
Burkholderia
functional homologs.
Chimeric hybrids between the
Pseudomonas
and
Burkholderia
homologs of RhlA illustrate selective modulation of rhamnolipid alkyl chain length production (blue arrows), implicating a putative cap-domain motif that covers the catalytic site and provides substrate specificity to the enzyme. This semi-rational mutant-based survey reveals promising ‘hot-spots’ for the modulation of rhamnolipid congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between these functional homologs.