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Title: Rhamnolipid and poly (hydrozyalkanoate) biosynthesis in 3-hydrozyacyl-ACP:COA transacylase (phaG) - knockouts of pseudomonas chloroaphis

Author
item Solaiman, Daniel
item Ashby, Richard - Rick
item Cross, Nicole
item Lai, Bun-Hong
item Zerkowski, Jonathan

Submitted to: Biocatalysis and Agricultural Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2013
Publication Date: 10/26/2013
Citation: Solaiman, D., Ashby, R.D., Crocker, N.V., Lai, B., Zerkowski, J.A. 2013. Rhamnolipid and poly (hydrozyalkanoate) biosynthesis in 3-hydrozyacyl-ACP:COA transacylase (phaG) - knockouts of pseudomonas chloroaphis. Biocatalysis and Agricultural Biotechnology. DOI: 10.1016/j.bcab.2013.09.009.

Interpretive Summary: Rhamnolipids (RLs) are biobased surfactants produced by some bacteria. RL is environmentally friendly because it is biodegradable; it is “green” because it can be produced in fermenter; and its production is sustainable because non-petroleum-based renewable feedstocks are used as feedstocks. RL has high commercial value because it can be used as active ingredient in washing solutions and cleaning products, as biopesticide for controlling fungal molds and algal blooms, as antibiotic for retarding the growth of bacteria, and as an adjuvant to promote wound healing. An opportunistic human pathogenic bacterium (called Pseudomonas aeruginosa) is currently the most widely used organism for industrial RL production. ARS had developed and received a patent for a process that uses a non-pathogenic bacterium (called Pseudomonas chlororaphis) to make RL. However, it is important that the RL and related metabolic flows of this organism is clearly understood in order to carry out knowledge-based optimization of production to lower cost. In the present work, we characterized the synthesis of a biodegradable polymer (called polyhydroxyalkanoate or PHA) in P. chlororaphis. We also cloned, characterized, and inactivate a gene called phaG that was thought to suck a common starting material away from RL synthesis to fuel PHA production instead. Our study has produced important information regarding the interplay of RL and PHA production in P. chlororaphis, laying the groundwork for future research to increase the production of RL in this non-pathogenic organism in a sensible manner.

Technical Abstract: The 3-hydroxyacyl-ACP:CoA transacylase gene (phaG(Pc30761)) of P. chlororaphis NRRL B-30761 was cloned and analyzed. The nucleotide and translated amino-acid sequences of phaG(Pc30761) had 99% identities (at 100% query coverage) with the phaG gene of P. fluorescens O6. Two phaG-knockout strains of P. chlororaphis (i.e., phaG::Tn5(#1) and (#5)) were constructed using an oligo-mediated recombineering technique mediated by recombinases recT and recE of P. syringe. As with the parental NRRL B-30761 strain, the phaG::Tn5(#1) and (#5) knockouts produced mono-rhamnolipids (predominantly the Rh1-C10-C12:1 and Rh1-C10-C12:0 congeners) at a similar crude yields of 0.68 – 0.87 g/L under the present experimental conditions. Under R1L-producing conditions without shaking, P. chlororaphis NRRL B-30761 produced small amount of medium-chain-length poly(hydroxyalkanoate)(PHAMCL) detectable only in lyophilized whole cells by GC/MS, which showed the repeat-units of the PHAMCL as predominantly b-hydroxydecanoate (C10:0; 37 mol%), b-hydroxydodecanoate (C12:0; 28 mol%), and b-hydroxydodecenoate (C12:1; 31 mol%). Similar results were observed with the phaG::Tn5(#1) and (#5) knockout strains. We also studied PHA production under shake-flask fermentation condition with 200-250 rpm rotary shaking. The parental and the 2 phaG knockouts produced only small amount of PHAMCL when grown on glucose or gluconate. GC/MS data showed that the PHAMCL of all three strains grown on gluconate contained predominantly the C10:0 (39 mol%) and C12:0 (52 mol%) repeat-units. When grown on oleic acid, however, all three strains produced sufficient amounts of PHAMCL to allow collection by precipitation in cold methanol. The polymers obtained from all three strains contained C8:0 (35 mol%), C10:0 (26 mol%), C14:1 (20 mol%), and C12:0 (13 mol%) as the predominant repeat-units.