Wax ester production in Rhodococus

Rhodococcus is a genus of soil bacteria that are among the best-studied oleaginous bacteria, and have considerable potential for the sustainable production of lipid-based chemicals. Herein, I characterized the biosynthesis of wax esters (WEs) in Rhodococcus jostii RHA1 and created tools to develop the biocatalytic potential of rhodococci. In Section 3.1, I established that RHA1 produces WEs and identified key enzymes involved in this production. Specifically, RHA1 produced WEs to 0.0002% of cellular dry weight (CDW) during exponential growth on glucose. These WEs contained 31 to 34 carbon atoms and were saturated. Bioinformatics revealed that RHA1 contains a putative fatty acyl-CoA reductase (FcrA). Purified FcrA catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45±3 nmol/mg∙min and dodecanal to dodecanol with a specific activity of 5300±300 nmol/mg∙min. A strain of RHA1 overproducing FcrA accumulated WEs to ~13% CDW. In Section 3.2, I expanded the genetic tools available in rhodococci, creating pSYN, a modular integrative-vector. I employed this vector to identify and characterize P₁₀, a strong, potentially constitutive rhodococcal promoter. Various strength promoters were created from P₁₀, resulting in the PT₂, PT₁, and PM₆ promoters, which were 1.3-, 2.2-, and 6-fold stronger, respectively, than Pnit, a constitutive promoter previously characterized in Rhodococcus. RHA1 transformed with a single copy of fcrA under the control of these various promoters accumulated WEs. In Section 3.3, I further developed RHA1 as a WE biocatalyst. Screening a variety of enzymes identified WS2 of Marinobacter hydrocarbonoclasticus DSM 8798 as an effective wax synthase in RHA1. Cassettes for the co-expression of chromosomally integrated fcrA and ws2 were created and transformed into RHA1; resulting in a biocatalyst that accumulated WEs to greater than 15% CDW, at yields of 0.05 g/g glucose, while maintaining 80% of the specific growth rate of WT. Accumulated WEs were 29 to 38 carbon atoms in length, of which 75% were unsaturated, with a ~2:1 mix of mono- and diunsaturated species. Overall, this thesis provides insight into the biosynthesis of WEs in rhodococci and facilitates the development of this genus for biocatalytic applications, including the production of high-value neutral lipids.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat