A biocatalyst for sustainable wax ester production: re-wiring lipid accumulation in Rhodococcus to yield high-value oleochemicals

Abstract

Oleaginous microorganisms, such as those found in the Rhodococcus genus, have considerable potential for the sustainable production of lipid-based chemicals. Herein, we rewired lipid accumulation in Rhodococcus jostii RHA1 to create an industrially viable biocatalyst for the production of high-value wax esters (WEs). To efficiently manipulate these non-model bacteria, we first expanded the genetic tools available in rhodococci, creating pSYN, an integrative, modular expression vector. We employed this vector to screen predicted promoters, creating a library of strong constitutive promoters. RHA1 strains with a chromosomal insertion of fcrA, encoding a fatty acyl-CoA reductase, under the control of constitutive promoters accumulated WEs. We next screened wax synthases, identifying WS2 of Marinobacter hydrocarbonoclasticus DSM 8798 as the most effective at increasing WE levels in RHA1. Cassettes for the co-expression of chromosomally integrated fcrA and ws2 were created and transformed into RHA1, yielding a biocatalyst that, when grown in flasks, accumulated WEs to greater than 15% CDW, at yields of 0.05 g per g glucose, while maintaining 80% of the specific growth rate of WT. The 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. In fed-batch fermentations, the biocatalyst produced WEs with a titer, rate, and yield of approximately 5 g L⁻¹, 1 g L⁻¹ day⁻¹, and 0.025 g per g glucose, respectively. Overall, these results highlight the potential of Rhodococcus for the sustainable production of high-value unsaturated WEs, and facilitate the development of this genus for biocatalytic applications.