title: Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels.
abstract: BACKGROUND
  Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations.
  
  
  RESULTS
  An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L(-1) glycerol, produced 2.3 g L(-1) of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L(-1)) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L(-1) glycerol, 40 g L(-1) glucose, and 40 g L(-1) xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L(-1) of TAGs, representing 51.2% of the CDM. In addition, when 20 g L(-1) glycerol was pulse-loaded into the culture with 40 g L(-1) glucose and 40 g L(-1) xylose at the stationary growth phase, MITGM-173 produced 14.3 g L(-1) of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L(-1) glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-1) on the pulse addition of glycerol.
  
  
  CONCLUSIONS
  We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kurosawa
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA ; Present address: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems Biotechnology, Forschungszentrum Juelich, 52425 Juelich, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Radek
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
authors.forename: Jens K
authors.initials: JK
authors.lastname: Plassmeier
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA ; Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Sinskey
keywords: Adaptive evolution; Co-fermentation; Glycerol utilization; Renewable fuels; Rhodococcus opacus; Triacylglycerol