Rhodococcus spp. strains are widespread in diverse natural and anthropized environments thanks to their high metabolic versatility, biodegradation activities, and unique adaptation capacities to several stress conditions such as the presence of toxic compounds and environmental fluctuations. Additionally, the capability of Rhodococcus spp. strains to produce high value-added products has received considerable attention, mostly in relation to lipid accumulation. In relation with this, several works carried out omic studies and genome comparative analyses to investigate the genetic and genomic basis of these anabolic capacities, frequently in association with the bioconversion of renewable resources and low-cost substrates into triacylglycerols. This review is focused on these omic analyses and the genetic and metabolic approaches used to improve the biosynthetic and bioconversion performance of Rhodococcus. In particular, this review summarizes the works that applied heterologous expression of specific genes and adaptive laboratory evolution approaches to manipulate anabolic performance. Furthermore, recent molecular toolkits for targeted genome editing as well as genome-based metabolic models are described here as novel and promising strategies for genome-scaled rational design of Rhodococcus cells for efficient biosynthetic processes application.

Department of Pharmacy and Biotechnology University of Bologna Via Irnerio 42 40126 Bologna Italy

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Alvarez HM, Herrero OM, Silva RA, Hernández MA, Lanfranconi MP, Villalba MS. Insights into the metabolism of oleaginous Rhodococcus spp. Appl Environ Microbiol. 2019;85(18):1–12. doi: 10.1128/AEM.00498-19. PubMed DOI PMC

Alvarez HM, Silva RA, Herrero M, Hernández MA, Villalba MS. Metabolism of triacylglycerols in Rhodococcus species: insights from physiology and molecular genetics. J Mol Biochem. 2013;2:2119–2130. doi: 10.1007/s00253-012-4360-1. DOI

Anthony WE, Carr RR, Delorenzo DM, et al. Development of Rhodococcus opacus as a chassis for lignin valorization and bioproduction of high-value compounds. Biotechnol Biofuels. 2019;12(1):1–14. doi: 10.1186/s13068-019-1535-3. PubMed DOI PMC

Auffret M, Labbé D, Thouand G, Greer CW, Fayolle-Guichard F. Degradation of a mixture of hydrocarbons, gasoline, and diesel oil additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis. Appl Environ Microbiol. 2009;75(24):7774–7782. doi: 10.1128/AEM.01117-09. PubMed DOI PMC

Bell KS, Philp JC, Aw DWJ, Christofi N. A review: the genus Rhodococcus. J Appl Microbiol. 1998;85(2):195–210. doi: 10.1046/j.1365-2672.1998.00525.x. PubMed DOI

Bugg TDH. Oxygenases: mechanisms and structural motifs for O2 activation. Curr Opin Chem Biol. 2001;5(5):550–555. doi: 10.1016/S1367-5931(00)00236-2. PubMed DOI

Busch H, Hagedoorn PL, Hanefeld U. Rhodococcus as a versatile biocatalyst in organic synthesis. Int J Mol Sci. 2019;20(19):1–36. doi: 10.3390/ijms20194787. PubMed DOI PMC

Cai C, Xu Z, Xu M, Cai M, Jin M. Development of a Rhodococcus opacus cell factory for valorizing lignin to muconate. ACS Sustain Chem Eng. 2020;8(4):2016–2031. doi: 10.1021/acssuschemeng.9b06571. DOI

Cappelletti M, Fedi S, Honda K, Ohtake H, Turner R, Zannoni D (2010) Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene. J Biotechnol 150:259. 10.1016/j.jbiotec.2010.09.150

Cappelletti M, Fedi S, Zampolli J, et al. Phenotype microarray analysis may unravel genetic determinants of the stress response by Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7. Res Microbiol. 2016;167(9–10):766–773. doi: 10.1016/j.resmic.2016.06.008. PubMed DOI

Cappelletti M, Pinelli D, Fedi S, Zannoni D, Frascari D (2018) Aerobic co-metabolism of 1,1,2,2-tetrachloroethane by Rhodococcus aetherivorans TPA grown on propane: kinetic study and bioreactor configuration analysis . J Chem Technol Biotechnol 93(1):155–165. 10.1002/jctb.5335

Cappelletti M, Presentato A, Piacenza E, Firrincieli A, Turner RJ, Zannoni D. Biotechnology of Rhodococcus for the production of valuable compounds. Appl Microbiol Biotechnol. 2020;104(20):8567–8594. doi: 10.1007/s00253-020-10861-z. PubMed DOI PMC

Cappelletti M, Presentato A, Milazzo G et al (2015) Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes. Front Microbiol 6:1–15. 10.3389/fmicb.2015.00393 PubMed PMC

Cappelletti M, Zampolli J, Di Gennaro P, Zannoni D (2019) Genomics of Rhodococcus. In: Alvarez HM (ed) Biology of Rhodococcus second edition of the series Microbiology monographs. Springer, Heidelberg, p. 23–60. 10.1007/978-3-030-11461-9

Ceniceros A, Dijkhuizen L, Petrusma M, Medema MH. Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus. BMC Genomics. 2017;18(1):1–16. doi: 10.1186/s12864-017-3966-1. PubMed DOI PMC

De Carvalho CCCR, Costa SS, Fernandes P, Couto I, Viveiros M (2014) Membrane transport systems and the biodegradation potential and pathogenicity of genus Rhodococcus. Front Physiol 5:1–13. 10.3389/fphys.2014.00133 PubMed PMC

DeLorenzo DM, Henson WR, Moon TS. Development of chemical and metabolite sensors for Rhodococcus opacus PD630. ACS Synth Biol. 2017;6:1973–1978. doi: 10.1021/acssynbio.7b00192. PubMed DOI

DeLorenzo DM, Moon TS. Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630. ACS Synth Biol. 2019;8:1921–1930. doi: 10.1021/acssynbio.9b00213. PubMed DOI

DeLorenzo DM, Rottinghaus AG, Henson WR, Moon TS. Molecular toolkit for gene expression control and genome modification in Rhodococcus opacus PD630. ACS Synth Biol. 2018;7:727–738. doi: 10.1021/acssynbio.7b00416. PubMed DOI

Desomer J, Dhaese P, Van Montagu M (1990) Transformation of Rhodococcus fascians by high-voltage electroporation and development of R. fascians cloning vectors. Appl Environ Microbiol 56(9):2818–2825. 10.1128/aem.56.9.2818-2825.1990 PubMed PMC

Dragosits M, Mattanovich D. Adaptive laboratory evolution - principles and applications for biotechnology. Microb Cell Fact. 2013;12(1):1–17. doi: 10.1186/1475-2859-12-64. PubMed DOI PMC

Duran R (1998) New shuttle vectors for Rhodococcus sp. R312 (formerly Brevibacterium sp. R312), a nitrile hydratase producing strain. J Basic Microbiol 38(2):101–106 PubMed

Gatti DL, Palfey BA, Lah MS, Entsch B, Massey V, Ballou DP, Ludwig ML (1994) The mobile flavin of 4-OH benzoate hydroxylase. Science 266(5182):110–4. 10.1126/science.7939628 PubMed

van der Geize R, Dijkhuizen L. Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Curr Opin Microbiol. 2004;7(3):255–261. doi: 10.1016/j.mib.2004.04.001. PubMed DOI

Guevara G, Flores YO, De Las Heras LF, Perera J, Navarro Llorens JM (2019) Metabolic engineering of Rhodococcus ruber Chol-4: a cell factory for testosterone production. PLoS One 14(7). 10.1371/journal.pone.0220492 PubMed PMC

Henson WR, Campbell T, DeLorenzo DM, Gao Y, Berla B, Kim SJ, Foston M, Moon TS, Dantas G. Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus. Metab Eng. 2018;49:69–83. doi: 10.1016/j.ymben.2018.06.009. PubMed DOI

Hernández MA, Alvarez HM. Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii. Microbiol (united Kingdom) 2019;165(1):4–14. doi: 10.1099/mic.0.000736. PubMed DOI

Hernández MA, Arabolaza A, Rodríguez E, Gramajo H, Alvarez HM. The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630. Appl Microbiol Biotechnol. 2013;97(5):2119–2130. doi: 10.1007/s00253-012-4360-1. PubMed DOI

Hernández MA, Comba S, Arabolaza A, Gramajo H, Alvarez HM. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains. Appl Microbiol Biotechnol. 2015;99(5):2191–2207. doi: 10.1007/s00253-014-6002-2. PubMed DOI

Hernández MA, Gleixner G, Sachse D, Alvarez HM (2017) Carbon allocation in Rhodococcus jostii RHA1 in response to disruption and overexpression of nlpR regulatory gene, based on 13C-labeling analysis. Front Microbiol 8:1–11. 10.3389/fmicb.2017.01992 PubMed PMC

Hernández MA, Mohn WW, Martínez E, Rost E, Alvarez AF, Alvarez HM. Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved in their metabolism. BMC Genomics. 2008;9:1–13. doi: 10.1186/1471-2164-9-600. PubMed DOI PMC

Hetzler S, Steinbüchel A. Establishment of cellobiose utilization for lipid production in Rhodococcus opacus PD630. Appl Environ Microbiol. 2013;79:3122–3125. doi: 10.1128/AEM.03678-12. PubMed DOI PMC

Huang L, Zhao L, Zan X, Song Y, Ratledge C. Boosting fatty acid synthesis in Rhodococcus opacus PD630 by overexpression of autologous thioesterases. Biotechnol Lett. 2016;38(6):999–1008. doi: 10.1007/s10529-016-2072-9. PubMed DOI

Hwangbo M, Chu KH (2020) Recent advances in production and extraction of bacterial lipids for biofuel production. Sci Total Environ 734. 10.1016/j.scitotenv.2020.139420 PubMed

Ivshina IB, Tyumina EA, Kuzmina MV, Vikhareva EV. Features of diclofenac biodegradation by Rhodococcus ruber IEGM 346. Sci Rep. 2019;9(1):1–13. doi: 10.1038/s41598-019-45732-9. PubMed DOI PMC

Jiao S, Yu H, Shen Z. Core element characterization of Rhodococcus promoters and development of a promoter-RBS mini-pool with different activity levels for efficient gene expression. N Biotechnol. 2018;44:41–49. doi: 10.1016/j.nbt.2018.04.005. PubMed DOI

Kalscheuer R, Arenskötter M, Steinbüchel A. Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids) Appl Microbiol Biotechnol. 1999;52(4):508–515. doi: 10.1007/s002530051553. PubMed DOI

Kim HM, Chae TU, Choi SY, Kim WJ, Lee SY. Engineering of an oleaginous bacterium for the production of fatty acids and fuels. Nat Chem Biol. 2019;15(7):721–729. doi: 10.1038/s41589-019-0295-5. PubMed DOI

Kis Á, Laczi K, Zsíros S et al (2017) Characterization of the Rhodococcus sp. MK1 strain and its pilot application for bioremediation of diesel oil-contaminated soil. Acta Microbiol Immunol Hung 64(4):463–482. 10.1556/030.64.2017.037 PubMed

Kis Á, Laczi K, Zsíros S, Rákhely G, Perei K. Biodegradation of animal fats and vegetable oils by Rhodococcus erythropolis PR4. Int Biodeterior Biodegrad. 2015;105:114–119. doi: 10.1016/j.ibiod.2015.08.015. DOI

Kurosawa K, Laser J, Sinskey AJ. Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcusopacus to lignocellulose-derived inhibitors. Biotechnol Biofuels. 2015;8:76. doi: 10.1186/s13068-015-0258-3. PubMed DOI PMC

Kurosawa K, Plassmeier J, Kalinowski J, Rückert C, Sinskey AJ. Engineering L-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production. Metab Eng. 2015;30:89–95. doi: 10.1016/j.ymben.2015.04.006. PubMed DOI

Kurosawa K, Radek A, Plassmeier JK, Sinskey AJ. Improved glycerol utilization by a triacylglycerol-producing Rhodococcusopacus strain for renewable fuels. Biotechnol Biofuels. 2015;8:31. doi: 10.1186/s13068-015-0209-z. PubMed DOI PMC

Kurosawa K, Wewetzer SJ, Sinskey AJ. Engineering xylose metabolism in triacylglycerol-producing Rhodococcusopacus for lignocellulosic fuel production. Biotechnol Biofuels. 2013;6:134. doi: 10.1186/1754-6834-6-134. PubMed DOI PMC

Kurosawa K, Wewetzer SJ, Sinskey AJ. Triacylglycerol production from corn stover using a xylose-fermenting Rhodococcusopacus strain for lignocellulosic biofuels. J Microbial Biochem Technol. 2014;6:254–259. doi: 10.4172/1948-5948.1000153. DOI

Laczi K, Kis Á, Horváth B, et al. Metabolic responses of Rhodococcus erythropolis PR4 grown on diesel oil and various hydrocarbons. Appl Microbiol Biotechnol. 2015;99(22):9745–9759. doi: 10.1007/s00253-015-6936-z. PubMed DOI

Larcher S, Yargeau V. Biodegradation of sulfamethoxazole by individual and mixed bacteria. Appl Microbiol Biotechnol. 2011;91(1):211–218. doi: 10.1007/s00253-011-3257-8. PubMed DOI

Larkin MJ, Kulakov LA, Allen CCR (2005) Biodegradation and Rhodococcus - masters of catabolic versatility. Curr Opin Biotechnol 16(3 SPEC. ISS.):282–290. 10.1016/j.copbio.2005.04.007 PubMed

Liang Y, Jiao S, Wang M, Yu H, Shen Z. A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH. Metab Eng. 2020;57:13–22. doi: 10.1016/j.ymben.2019.10.003. PubMed DOI

MacEachran DP, Sinskey AJ. The Rhodococcus opacus TadD protein mediates triacylglycerol metabolism by regulating intracellular NAD(P)H pools. Microb Cell Fact. 2013;12(1):1. doi: 10.1186/1475-2859-12-104. PubMed DOI PMC

Mandal B, Prabhu A, Pakshirajan K, Veeranki Dasu V. Construction and parameters modulation of a novel variant Rhodococcus opacus BM985 to achieve enhanced triacylglycerol-a biodiesel precursor, using synthetic dairy wastewater. Process Biochem. 2019;84(June):9–21. doi: 10.1016/j.procbio.2019.05.031. DOI

Martínková L, Uhnáková B, Pátek M, Nešvera J, Křen V. Biodegradation potential of the genus Rhodococcus. Environ Int. 2008;35(1):162–177. doi: 10.1016/j.envint.2008.07.018. PubMed DOI

Mitani Y, Nakashima N, Sallam KI, Toriyabe T, Kondo K, Tamura T. Advances in the development of genetic tools for the genus Rhodococcus. Actinomycetologica. 2006;20(2):55–61. doi: 10.3209/saj.20.55. DOI

O'Brien EJ, Monk JM, Palsson BO. Using genome-scale models to predict biological capabilities. Cell. 2015;161:971–987. doi: 10.1016/j.cell.2015.05.019. PubMed DOI PMC

Orro A, Cappelletti M, D’Ursi P et al (2015) Genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: genetic determinants and metabolic abilities with environmental relevance. PLoS One 10(10):1–41. 10.1371/journal.pone.0139467 PubMed PMC

Pátek M, Grulich M, Nešvera J. Stress response in Rhodococcus strains. Biotechnol Adv. 2021 doi: 10.1016/j.biotechadv.2021.107698. PubMed DOI

Presentato A, Cappelletti M, Sansone A et al (2018a) Aerobic growth of Rhodococcus aetherivorans BCP1 using selected naphthenic acids as the sole carbon and energy sources. Front Microbiol 9(APR):1–15. 10.3389/fmicb.2018.00672 PubMed PMC

Presentato A, Piacenza E, Anikovskiy M, Cappelletti M, Zannoni D, Turner RJ. Rhodococcus aetherivorans BCP1 as cell factory for the production of intracellular tellurium nanorods under aerobic conditions. Microb Cell Factories. 2016;15:204. doi: 10.1186/s12934-016-0602-8. PubMed DOI PMC

Presentato A, Piacenza E, Anikovskiy M, Cappelletti M, Zannoni D, Turner RJ. Biosynthesis of selenium-nanoparticles and -nanorods as a product of selenite bioconversion by the aerobic bacterium Rhodococcus aetherivorans BCP1. New Biotechnol. 2018;41:1–8. doi: 10.1016/j.nbt.2017.11.002. PubMed DOI

Presentato A, Piacenza E, Darbandi A, Anikovskiy M, Cappelletti M, Zannoni D, Turner RJ. Assembly, growth and conductive properties of tellurium nanorods produced by Rhodococcus aetherivorans BCP1. Sci Rep. 2018;8:3923. doi: 10.1038/s41598-018-22320-x. PubMed DOI PMC

Presentato A, Piacenza E, Turner RJ, Zannoni D, Cappelletti M. Processing of metals and metalloids by actinobacteria: cell resistance mechanisms and synthesis of metal(loid)-based nanostructures. Microorganisms. 2020;8(12):1–37. doi: 10.3390/microorganisms8122027. PubMed DOI PMC

Roell GW, Carr RR, Campbell T, et al. A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630. Metab Eng. 2019;55(June):120–130. doi: 10.1016/j.ymben.2019.06.013. PubMed DOI

Sandberg TE, Salazar MJ, Weng LL, Palsson BO, Feist AM. The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology. Metab Eng. 2019;56(August):1–16. doi: 10.1016/j.ymben.2019.08.004. PubMed DOI PMC

Schweizer E, Hofmann J. Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiol Mol Biol Rev. 2004;68(3):501–517. doi: 10.1128/MMBR.68.3.501-517.2004. PubMed DOI PMC

Sekizaki T, Tanoue T, Osaki M, Shimoji Y, Tsubaki S, Takai S. Improved electroporation of Rhodococcus equi. J Vet Med Sci. 1998;60(2):277–279. doi: 10.1292/jvms.60.277. PubMed DOI

Shao Z, Dick WA, Behki RM (1995) An improved Eschehchia coli‐Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation. Lett Appl Microbiol 21(4):261–266. 10.1111/j.1472-765X.1995.tb01056.x PubMed

Singer ME, Finnerty WR. Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. J Bacteriol. 1988;170(2):638–645. doi: 10.1128/jb.170.2.638-645.1988. PubMed DOI PMC

Spence EM, Calvo-Bado L, Mines P, Bugg TDH. Metabolic engineering of Rhodococcus jostii RHA1 for production of pyridine-dicarboxylic acids from lignin. Microb Cell Fact. 2021;20(1):1–12. doi: 10.1186/s12934-020-01504-z. PubMed DOI PMC

Tajparast M, Frigon D. Genome-scale metabolic model of Rhodococcus jostii RHA1 (iMT1174) to study the accumulation of storage compounds during nitrogen-limited condition. BMC Syst Biol. 2015;9:43. doi: 10.1186/s12918-015-0190-y. PubMed DOI PMC

Tajparast M, Frigon D (2018) Predicting the accumulation of storage compounds by Rhodococcus jostii RHA1 in the feast-famine growth cycles using genome-scale flux balance analysis. PLoS One 13 PubMed PMC

Tyumina EA, Bazhutin GA, Vikhareva EV, Selyaninov AA, Ivshina IB (2019) Diclofenac as a factor in the change of Rhodococcus metabolism. IOP Conf Ser Mater Sci Eng 487(1). 10.1088/1757-899X/487/1/012027

Weidhaas JL, Chang DPY, Schroeder ED. Biodegradation of nitroaromatics and RDX by isolated Rhodococcus opacus. J Environ Eng. 2009;135(10):1025–1031. doi: 10.1061/(asce)ee.1943-7870.0000072. DOI

Xie S, Sun S, Lin F et al (2019) Mechanism-guided design of highly efficient protein secretion and lipid conversion for biomanufacturing and biorefining. Adv Sci 6(13). 10.1002/advs.201801980 PubMed PMC

Xiong X, Lian J, Yu X, Garcia-Perez M, Chen S. Engineering levoglucosan metabolic pathway in Rhodococcus jostii RHA1 for lipid production. J Ind Microbiol Biotechnol. 2016;43:1551–1560. doi: 10.1007/s10295-016-1832-9. PubMed DOI

Xiong X, Wang X, Chen S. Engineering of a xylose metabolic pathway in Rhodococcus spp. strains. Appl Environ Microbiol. 2012;78:5483–5491. doi: 10.1128/AEM.08022-11. PubMed DOI PMC

Xiong X, Wang X, Chen S. Engineering of an L-arabinose metabolic pathway in Rhodococcus jostii RHA1 for biofuel production. J Ind Microbiol Biotechnol. 2016;43:1017–1025. doi: 10.1007/s10295-016-1778-y. PubMed DOI

Yoneda A, Henson WR, Goldner NK, Park KJ, Forsberg KJ, Kim SJ, Pesesky MW, Foston M, Dantas G, Moon TS. Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630. Nucleic Acids Res. 2016;44:2240–2254. doi: 10.1093/nar/gkw055. PubMed DOI PMC

Transcriptomic Analysis of the Dual Response of Rhodococcus aetherivorans BCP1 to Inorganic Arsenic Oxyanions

The Complete Genome Sequence and Structure of the Oleaginous Rhodococcus opacus Strain PD630 Through Nanopore Technology