|Ashby, Richard - Rick|
Submitted to: American Oil Chemists' Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 11/10/2014
Publication Date: 4/1/2015
Citation: Solaiman, D., Ashby, R.D. 2015. Genetically engineered rhamnolipid-producing organism for glycerol utilization [abstract]. American Oil Chemists' Society Meeting. p. 5.
Technical Abstract: Rhamnolipid (RL) is a microbial glycolipid currently developed for industrial use as a biobased surfactant. It also possesses antimicrobial activity that is attractive for applications in sanitizing washes. Glycerol byproduct stream from biodiesel production is a promising low-cost substrate for microbial production of biobased chemicals. P. chlororaphis is a non-pathogenic organism used for RL production; however, it cannot readily use glycerol in fermentation. In this paper, we report the cloning and expression of E. coli glycerol-utilization genes into P. chlororaphis to overcome its shortcoming in using glycerol as a growth substrate. Two genes, glycerol facilitator (glpF) and glycerol kinase (glpK), were PCR-amplified from an E. coli K12 host. They were spliced, either individually or together, downstream from a Pseudomonas promoter (P2) in the expression vector pBS29-P2-gfp using In-Fusion method. Positive clones were identified by microplate high-throughput fluorescence screening and then confirmed by plasmid screening. The growth of P. chlororaphis transformants were compared using glycerol as a sole substrate. The results showed that concomitant expression of glpF and glpK in recombinant P. chlororaphis caused faster growth than the wild-type and the transformants expressing individual glpF or glpK. The final glycerol consumption and cell density, however, were similar in all tested organisms, indicating that glpF and glpK together govern the kinetics of the initial steps of glycerol metabolism. HPLC-ELSD analysis showed that the synthesis of RL was not affected by the expression of the heterologous genes. The results lay the groundwork for further improvement of glycerol utilization in P. chlororaphis through metabolic engineering of the subsequent steps in the pathway.