TY - JOUR
T1 - Chloroplast engineering of the green microalgae Chlamydomonas reinhardtii for the production of HAA, the lipid moiety of rhamnolipid biosurfactants
AU - Miró-Vinyals, Bernat
AU - Artigues, Margalida
AU - Wostrikoff, Katia
AU - Monte, Elena
AU - Broto-Puig, Francesc
AU - Leivar, Pablo
AU - Planas, Antoni
N1 - Funding Information:
This work was supported by the Spanish Ministry of Science and Innovation (MICINN) [grant PID2019–104350RB-I00 ] (to A.P.); the AGAUR agency of Generalitat de Catalunya [grant 2017SGR-727 ] (to A.P.); and the Ramon Llull University / Obra Social la Caixa Grants (to P.L.). Work at UMR7141 in Paris was funded through the “ Initiative d′Excellence” program (Grant “ DYNAMO ,” ANR-11-LABX-0011–01 ). Work at CRAG was funded by the FEDER / Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación [Grants PGC2018–099987-B-I00 and PID2021–122288NB-I00 ], the CERCA Programme / Generalitat de Catalunya [Grant 2017SGR-718 ], and the Spanish Ministry of Economy and Competitiveness / “Severo Ochoa Programme for Centres of Excellence in R&D ” [Grant CEX2019–000902-S ] (to E.M.). B.M-V. was recipient of a fellowship from the Department of Research and Universities of the Catalan Government and the European Social Fund ( 2018FI_B00823 ). We thank Guillaume Allorent and Giovanni Finazzi (CNRS-CEA, Grenoble) for hosting P.L. for a short stay and for materials transfer, Matthieu Mustas (IBCP, Paris) for experimental support on microbombardment, and Gisela Querol for lipid analysis.
Funding Information:
This work was supported by the Spanish Ministry of Science and Innovation (MICINN) [grant PID2019–104350RB-I00] (to A.P.); the AGAUR agency of Generalitat de Catalunya [grant 2017SGR-727] (to A.P.); and the Ramon Llull University / Obra Social la Caixa Grants (to P.L.). Work at UMR7141 in Paris was funded through the “Initiative d′Excellence” program (Grant “DYNAMO,” ANR-11-LABX-0011–01). Work at CRAG was funded by the FEDER / Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación [Grants PGC2018–099987-B-I00 and PID2021–122288NB-I00], the CERCA Programme / Generalitat de Catalunya [Grant 2017SGR-718], and the Spanish Ministry of Economy and Competitiveness / “Severo Ochoa Programme for Centres of Excellence in R&D” [Grant CEX2019–000902-S] (to E.M.). B.M-V. was recipient of a fellowship from the Department of Research and Universities of the Catalan Government and the European Social Fund (2018FI_B00823). We thank Guillaume Allorent and Giovanni Finazzi (CNRS-CEA, Grenoble) for hosting P.L. for a short stay and for materials transfer, Matthieu Mustas (IBCP, Paris) for experimental support on microbombardment, and Gisela Querol for lipid analysis.
Publisher Copyright:
© 2023 The Authors
PY - 2023/9/25
Y1 - 2023/9/25
N2 - Hydroxyalkanoyloxyalkanoates (HAA) are lipidic surfactants with a number of potential applications, but more remarkably, they are the biosynthetic precursors of rhamnolipids (RL), which are preferred biosurfactants thanks to their excellent physicochemical properties, biological activities, and environmental biodegradability. Because the natural highest producer of RLs is the pathogenic bacterium Pseudomonas aeruginosa, important efforts have been dedicated to transfer production to heterologous non-pathogenic microorganisms. Unicellular photosynthetic microalgae are emerging as important hosts for sustainable industrial biotechnology due to their ability to transform CO2 efficiently into biomass and bioproducts of interest. Here, we have explored the potential of the eukaryotic green microalgae Chlamydomonas reinhardtii as a chassis to produce RLs. Chloroplast genome engineering allowed the stable functional expression of the gene encoding RhlA acyltransferase from P. aeruginosa, an enzyme catalyzing the condensation of two 3-hydroxyacyl acid intermediaries in the fatty acid synthase cycle, to produce HAA. Four congeners of varying chain lengths were identified and quantified by UHPLC-QTOF mass spectrometry and gas chromatography, including C10-C10 and C10-C8, and the less abundant C10-C12 and C10-C6 congeners. HAA was present in the intracellular fraction, but also showed increased accumulation in the extracellular medium. Moreover, HAA production was also observed under photoautotrophic conditions based on atmospheric CO2. These results establish that RhlA is active in the chloroplast and is able to produce a new pool of HAA in a eukaryotic host. Subsequent engineering of microalgal strains should contribute to the development of an alternative clean, safe and cost-effective platform for the sustainable production of RLs.
AB - Hydroxyalkanoyloxyalkanoates (HAA) are lipidic surfactants with a number of potential applications, but more remarkably, they are the biosynthetic precursors of rhamnolipids (RL), which are preferred biosurfactants thanks to their excellent physicochemical properties, biological activities, and environmental biodegradability. Because the natural highest producer of RLs is the pathogenic bacterium Pseudomonas aeruginosa, important efforts have been dedicated to transfer production to heterologous non-pathogenic microorganisms. Unicellular photosynthetic microalgae are emerging as important hosts for sustainable industrial biotechnology due to their ability to transform CO2 efficiently into biomass and bioproducts of interest. Here, we have explored the potential of the eukaryotic green microalgae Chlamydomonas reinhardtii as a chassis to produce RLs. Chloroplast genome engineering allowed the stable functional expression of the gene encoding RhlA acyltransferase from P. aeruginosa, an enzyme catalyzing the condensation of two 3-hydroxyacyl acid intermediaries in the fatty acid synthase cycle, to produce HAA. Four congeners of varying chain lengths were identified and quantified by UHPLC-QTOF mass spectrometry and gas chromatography, including C10-C10 and C10-C8, and the less abundant C10-C12 and C10-C6 congeners. HAA was present in the intracellular fraction, but also showed increased accumulation in the extracellular medium. Moreover, HAA production was also observed under photoautotrophic conditions based on atmospheric CO2. These results establish that RhlA is active in the chloroplast and is able to produce a new pool of HAA in a eukaryotic host. Subsequent engineering of microalgal strains should contribute to the development of an alternative clean, safe and cost-effective platform for the sustainable production of RLs.
KW - Rhamnolipid biosurfactants. HAA precursor. RhlA acyltransferase. Microalgae Chlamydomonas reinhardtii. Chloroplast transformation. Metabolic Engineering
UR - http://www.scopus.com/inward/record.url?scp=85152605814&partnerID=8YFLogxK
U2 - 10.1016/j.nbt.2023.03.005
DO - 10.1016/j.nbt.2023.03.005
M3 - Article
C2 - 37004923
AN - SCOPUS:85152605814
SN - 1871-6784
VL - 76
SP - 1
EP - 12
JO - New Biotechnology
JF - New Biotechnology
ER -