Semi‐rational evolution of the 3‐(3‐hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA to improve rhamnolipid production in Pseudomonas aeruginosa and Burkholderia glumae

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. Database Model data are available in the PMDB database under the accession number http://www.ncbi.nlm.nih.gov/protein/PM0081867.

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

“…The biosynthesis of rhamnolipids starts with the formation of HAA from two β-hydroxy fatty acids by the enzyme RhlA ( Déziel et al, 2003 ). The substrate spectra of different RhlA enzymes are responsible for the chain-length spectrum of the final biosurfactant ( Dulcey et al, 2019 ; Germer et al, 2020 ). Subsequently, rhamnose units are attached to the HAA by activity of RhlB and RhlC, respectively, to form mono- and di-rhamnolipids ( Ochsner et al, 1994 ; Rahim et al, 2001 ).…”

Heterologous Rhamnolipid Biosynthesis: Advantages, Challenges, and the Opportunity to Produce Tailor-Made Rhamnolipids

“…Such an approach using hybrid rhlAB operons from P. aeruginosa and B. glumae verified, that RhlA seems to mainly determine the fatty acid lengths used for the biosynthesis first of HAAs, but subsequent also for rhamnolipids . Similar experiments using the two originating species as hosts for such hybrid operons further indicated an influence of the host organism and its provided 3-hydroxyfatty acids (Dulcey et al, 2019).…”

“…In contrast, if the same operon was expressed in P. aeruginosa as host, the amount of C 12 -C 12 congener was only increased in a mixture with a still predominant C 10 -C 10 congener. It was further demonstrated, that mutagenized RhlA reached activities more than twice as much as the wild type RhlA (Dulcey et al, 2019) and amino acid exchanges of RhlB resulted in a shifted pattern of the rhamnolipid composition from C 10 -C 10 to C 10 -C 8 congeners indicating a further specificity of RhlB to specific HAAs containing certain fatty acid length (Han et al, 2014). Further enzyme modifications could result in a much more specific congener enriched composition through improved specificity of responsible enzymes to specific fatty acid chain lengths.…”

“…Another purpose for tailor-made rhamnolipids is to define the fatty acid chain lengths for each of the four species and possibly their degree of saturation customized for any type of specific applications. So far most of the publications used rhl genes from P. aeruginosa for the heterologous production of rhamnolipids in different hosts and only a few reports about the successful heterologous expression of rhl genes from Burkholderia Dulcey et al, 2019). However, it has been described for Pseudomonas desmolyticum to produce rhamnolipids with predominant C 6 -C 8 fatty acids (Jadhav et al, 2011), while other bacteria of the genus Thermus produce much longer rhamnolipids with up to 24 carbon atoms and a predominant C 16 -C 16 congener (Řezanka et al, 2011), but none of them were produced heterologously so far.…”

“…A further possibility to achieve novel chain lengths in rhamnolipids is to manipulate the involved enzymes by single amino acid or whole domain exchanges to alter the accepted length of 3-hydroxyfatty acids as it was recently done. Thereby, Dulcey et al (2019) created a chimeric RhlA variant using different original enzyme domains for RhlA originating form P. aeruginosa and B. glumae. Combined in an operon with rhlB from P. aeruginosa, the newly designed enzyme produced rhamnolipids with a predominant C 12 -C 12 congener when heterologous expressed in B. glumae, which is the average between the typical short-chain rhamnolipids from Pseudomnas and the long-chain rhamnolipids from Burkholderia.…”