A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

Abstract

Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 Ϯ 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 Ϯ 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon-or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ϳ13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C 17 versus C 16 ). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of highvalue neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.KEYWORDS fatty acyl-CoA reductase, lipid accumulation, Rhodococcus, wax esters, metabolic engineering M any bacterial species, particularly the mycolic acid-producing Actinobacteria, synthesize neutral lipids for energy storage. For example, the soil bacterium Rhodococcus jostii RHA1 (referred to as RHA1 here) accumulates neutral lipids up to 70% of cellular dry weight (CDW) in response to environmental stresses, such as nitrogen limitation (1-3). These neutral lipids are primarily triacylglycerides (TAGs) and are stored in lipid bodies within the cytoplasm (4). Proteins associated with these carbon storage organelles (5) facilitate the dynamic shuffling of lipids between utilization and storage, allowing the bacterium to sequester intracellular energy reserves as needed. Due in

Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species

“…For instance, Alvarez et al [54] demonstrated that R. opacus PD630 accumulated TAG as major inclusions together with some DAG and FFA, which constituted up to 76% and 87% of cellular dry weight when grown on gluconate or olive-oil, respectively, whereas R. rhodochrous accumulated both TAG and WE when cultured in media with glucose as the main carbon source [55]. In the experiments presented in the current study, TAG was identified as a major component of the lipid profiles of some of the sub-Antarctic species tested but there was no evidence of WE synthesis, as shown in other rhodococci species which make detectable but proportionately low quantities of WE [56]. However, TAG was a relatively small proportion of the total lipids detected in the sub-Antarctic strains, possibly because the growth conditions were not optimized for TAG synthesis.…”

Catabolism of Alkylphenols in Rhodococcus via a Meta-Cleavage Pathway Associated With Genomic Islands

“…However, alkylphenols may be funneled through ortho- cleavage by oxidizing the para -side chain to 4-hydroxyacetophenone or hydroquinone (Darby et al, 1987). Alternatively, oleaginous Rhodococcus strains such as RHA1 may be modified to use the Aph pathway to produce lipid-based commodity chemicals (e.g., Round et al, 2017) offering a method for valorization of alkylphenols via fatty-acid synthesis in this genus.…”

“…Rhodococcus is a genus of mycolic acid-producing Actinobacteria that catabolize a wide variety of aromatic compounds (Yam et al, 2010), including phenols (Kolomytseva et al, 2007; Gröning et al, 2014). These bacteria also have considerable potential as biocatalysts for the industrial production of compounds ranging from nitriles to steroids and high-value lipids (Alvarez et al, 1996; Round et al, 2017; Shields-Menard et al, 2017; Sengupta et al, 2019). In Rhodococcus , phenol catabolism is initiated by a two-component flavin-dependent monooxygenase (PheA1A2; Saa et al, 2010) to generate a catechol.…”

Steryl ester formation and accumulation in steroid-degrading bacteria

“…The organic phase was dried under nitrogen and lipids were suspended in 1 ml chloroform with 1% acetic acid. Aliquots were analyzed by GC-MS as described earlier for FAMEs and free cholesterol (62) and for wax esters (32). Cholesterol and fatty acid methyl-esters (FAMEs) were identified by comparison to authentic standards as well as matching against the NIST database (National Institute of Standards and Technology, v11).…”