Abstract
Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway’s overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway’s intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.
Highlights
Surfactants - surface active agents - are amphiphilic molecules, which tend to accumulate at the interface between polar and non-polar solvents
For the first time, the six genes from P. aeruginosa coding for the enzymes responsible for dTDP-Rha and mono-RLs biosynthesis were resynthesized according to codon usage of S. cerevisiae and functionally expressed in it, resulting in two final strains: dTDP-Rha producing strain Rhamnose Producing strain (RHP) and mono-RLs producing strain Rhamnolipid Producing strain (RLP)
A rhamnose producing strain was constructed because this compound is an important precursor of organic molecules and it is widely used in industrial and laboratorial scale applications, making it an interesting commercial product in addition to RLs
Summary
Surfactants - surface active agents - are amphiphilic molecules, which tend to accumulate at the interface between polar and non-polar solvents. Rhamnolipids are produced mainly by the pathogenic gram-negative bacterium Pseudomonas aeruginosa They are composed of one (mono-RLs) or two (di-RLs) rhamnose moieties linked to beta-hydroxy fatty acid chains that vary in number (1 to 3 chains), length (8 to 16 carbons) and degree of unsaturation[7]. Heterologous expression of P. aeruginosa genes involved in RLs biosynthesis in an alternative host would circumvent the regulatory network and the pathogenicity of natural producer. This may allow a more efficient production and reduced costs associated with downstream processes, thereby enhancing the economic feasibility of RLs production. Current strategies of microbial fermentation for RLs production is only economically feasible when they are required in the composition of high-priced products[10]
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