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Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

ABSTRACT: Rhamnolipids (RL) are well-studied biosurfactants naturally produced by pathogenic strains of P. aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however CH4 provides an intriguing alternative as a substrate for RL production because it is low-cost and may mitigate greenhouse gas emissions. Here we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum 20Z. RLs were inhibitory to M. alcaliphilum growth at low concentrations (<0.05 g/L), so adaptive evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/L of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared to the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 M of RLs, a 600-fold increase compared to the parent strain. This work has promise for developing technologies to produce fatty acid-derived bioporducts, including biosurfactants, from CH4.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Methylomicrobium Alcaliphilum (strain Dsm 19304 / Ncimb 14124 / Vkm B-2133 / 20z)

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD029605 | Pride | 2022-09-19

REPOSITORIES: Pride

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			Action	DRS
	R__24h_1.d.zip	Other
	R__24h_1__F019504_.mzid.gz	Mzid
	R__24h_1__F019504_.mzid_R__24h_1__F019504_.MGF	Mzid
	R__24h_2.d.zip	Other
	R__24h_2__F019505_.mzid.gz	Mzid

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Project description:Resistance of Saccharomyces cerevisiae to high furfural concentration is based on NADPH-dependent reduction by at least two oxireductases. Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural resistant strain and its parent in micro-aerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower furfural concentrations, NADH-dependent oxireductases are the main defence mechanism. At concentrations above 15 mM, however, [1-13C]-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through pentose-phosphate pathway increases and that NADPH-dependent oxireductases became the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is up-regulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterised ORF YKL071W and 4 further, likely NADPH-dependent oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inverse engineered significantly increased furfural resistance in the parent strain, thereby demonstrating these two enzymes to be key elements of the resistance phenotype. Experiment Overall Design: RNA levels were measured in glucose limited, micro-aerobic chemostat cultures with different concentrations of the growth inhibitor furfural. Two strains were compared: TMB3400-FT30-3 is a strain that has been evolutionary adapted to withstand high furfural concentrations. TMB3400 is its less resistant parent. Number of biological replicates: 2-3.

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Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

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