A BACTERIUM...
Engineered with an integrated Cre/loxP recombinase system, it allows gene insertion directly into the genome via simple plasmid electroporation, with successful integration easily selectable through fluorescent protein expression.
A TOOL...
That reconstructs the metabolic pathway needed to degrade a given input pollutant and suggests which genes to include in the plasmid for electroporation into Rhodococcus.
A BIOSENSOR...
To quantify high-value industrial products synthesized by our bacterium, such as triacylglycerols (TAGs), which can be tailored for biodiesel production.
Abstract
What if there were a bacterium that is easy to genetically modify, simple to reprogram metabolically, and capable of reporting its own production output?
BACMAN is a bacterial chassis built on three integrated components — a computational tool, a standardized host bacterium, and a biosensor — designed for applications in bioremediation and synthetic biology. It enables the rapid development of engineered strains capable of degrading pollutants while producing high-value biomolecules.
Our aim is to support researchers and practitioners by providing a system that assists in selecting which genes to insert and from which donor organisms, offers a well-characterized and modular host for gene integration, and incorporates a biosensor to detect key metabolic products — specifically triacylglycerols.
Why Rhodococcus?
The genus Rhodococcus represents a phylogenetically and catabolically diverse group of Gram-positive, non-motile, aerobic chemoorganotrophic bacteria, well-known for their oxidative metabolism and ability to utilize a wide variety of carbon and energy sources (Ludwig et al., 2012; Sangal et al., 2019). Members of this genus have been isolated from various extreme and contaminated environments, including polar, alpine, and marine regions, where their remarkable adaptability and metabolic plasticity allow them to survive and function efficiently (Kim et al., 2021; Gibu et al., 2019; Xiang et al., 2022).
The wide distribution of Rhodococcus spp. strains is due to their extraordinary metabolic versatility, which is comparable to that described only in a few other bacterial genera, and to their unique environmental persistence and robustness (LeBlanc et al. 2008; Cappelletti et al. 2016).This metabolic versatility is largely attributed to the presence of diverse catabolic genes, often acquired through horizontal gene transfer mediated by large plasmids (Kim et al., 2021). These genetic features enable Rhodococcus to degrade a wide range of both natural and xenobiotic compounds—such as aliphatic, aromatic (halogenated and nitro-substituted), heterocyclic and polycyclic aromatic hydrocarbons, alicyclic hydrocarbons, nitriles, cholesterol, and lignins (Kim et al., 2021).For instance, their metabolic potential is considered comparable to that of Pseudomonas species, placing Rhodococcus among the most biotechnologically relevant genera in environmental and industrial microbiology.
Rhodococcus strains have demonstrated particularly high efficiency in bioremediation and bioconversion of recalcitrant pollutants, including PAHs (Subashchandrabose et al., 2019), phenolic compounds (Barik et al., 2021), fungicides (Bai et al., 2017), pharmaceuticals (Ivshina et al., 2019), phthalates (Zhao et al., 2018), steroids (Ye et al., 2019), dyes (Maniyam et al., 2020a), and even heavy metals (Goswami et al., 2017). This efficiency is a consequence of both their enzymatic diversity and their tolerance to harsh and toxic conditions (Adenan et al., 2020; Dobrowolski et al., 2017; Yang et al., 2018; Zhao et al., 2021).
Moreover, as actinomycetes, Rhodococcus belongs to a broader class of microorganisms with a well-documented role in eco-friendly bioremediation strategies (Alvarez et al., 2017; Aparicio et al., 2018; Rathore et al., 2021; Usmani et al., 2021). Their natural occurrence in contaminated soils, groundwater, and boreholes further supports their application in real-world remediation efforts (Kim et al., 2018; Gibu et al., 2019).
Beyond environmental applications, Rhodococcus has proven valuable in industrial biotechnology. They are capable of producing biosurfactants useful in the oil, pharmaceutical, cosmetic, and food industries (Cappelletti et al., 2020; Kuyukina and Ivshina, 2010b), bioflocculants with low human and environmental toxicity (Cappelletti et al., 2020), enzymes (Krivoruchko et al., 2019), and pigments such as carotenoids with potential antimicrobial properties (Cappelletti et al., 2020; Çobanoğlu and Yazici, 2022; Madhukar, 2021). Additionally, Rhodococcus strains can assist in nitrogen fixation and pathogen suppression in agricultural soils, contributing to increased crop productivity (Joshi et al., 2019; Madhukar, 2021; Pirog et al., 2020; Ward et al., 2018).
These studies suggest the great role of the Rhodococcus genus as a promising chassis for applications in bioremediation, biosurfactant production, lignin valorization, and the bioproduction of high-value compounds.
Superpower of Rhodococcus Opacus PD630
In general Rhodococcus opacus PD630 is a metabolically robust and genetically accessible actinobacterium with considerable promise as a biotechnological chassis, particularly in the context of sustainable lipid and carotenoid production. Notably, this strain can accumulate triacylglycerols (TAGs) up to 87% of its dry cell weight under nitrogen-limited, carbon-rich conditions (Kurosawa et al., 2010), making it a powerful candidate for biodiesel and oleochemical production. In parallel, R. opacus PD630 has demonstrated significant potential in the biosynthesis of carotenoids—valuable pigments with antioxidant, antimicrobial, and industrial relevance. Through metabolic engineering and pathway optimization, various studies have successfully enhanced its native carotenoid production, and introduced heterologous pathways to produce specific molecules such as lycopene, β-carotene, and astaxanthin (Kurosawa et al., 2020; Holder et al., 2021). Moreover, carotenoid biosynthesis in R. opacus is compatible with its high TAG accumulation, enabling dual production of lipids and pigments in the same process (Kurosawa et al., 2020). This dual functionality increases its economic attractiveness for integrated biorefineries.
The strain’s ability to metabolize a broad range of carbon sources, including lignin-derived aromatics (phenol, vanillate, p-coumarate), via the β-ketoadipate pathway (Kurosawa et al., 2015), allows valorization of lignocellulosic waste into both lipids and carotenoids. Its tolerance to high substrate concentrations (up to 300 g/L glucose) and resistance to toxic intermediates further supports industrial scalability (Kurosawa et al., 2010; Chen et al., 2023). Synthetic biology tools—including inducible promoters, metabolite-responsive sensors, CRISPRi-based repression, and recombinase-mediated genome engineering—are increasingly available for PD630, enabling precise control of metabolic fluxes toward desired compounds (Holder et al., 2021). Altogether, the combination of high lipid accumulation, carotenoid biosynthetic capacity, broad catabolic flexibility, and growing genetic toolkits firmly establishes R. opacus PD630 as a versatile and valuable chassis for biotechnological applications, ranging from biofuels and high-value chemicals to functional food and cosmetics.
Managing rhodococcus genome with HERO
The goal of our wetlab work is to engineer Rhodococcus opacus PD630 as a chassis that can be easily modified for transgene insertion, using the Cre/Lox recombinase system.
The construct will be inserted into a low-transcribed, non-regulatory region of the genome. We will also build a modular plasmid to facilitate the integration of genes of interest. Finally, we aim to make the Rhodococcus opacus PD630 colonies more visually distinguishable.
C. Geni per la ricombinasi Cre e la sequenza loxP sono inseriti in un locus del genoma di PD630 che non ha funzioni essenziali.
D. Utilizzo del sistema Cre/loxP già integrato nel genoma per facilitare l’ingegnerizzazione del batterio.
Managing pathways prediction with Spiderman
We are developing a user-friendly platform to support researchers in employing our chassis. The system takes a pollutant to degrade as input, constructs the required metabolic pathways, and finally provides suggestions for which genes to insert into the plasmid — including the corresponding sequences. The pathways are designed to lead to metabolites already native to Rhodococcus opacus PD630, and each reaction step is linked to an enzyme selected via its EC number. As a final output, the platform returns the recombination-ready sequences.
Managing TAGs detection with Ironman
To quantify the high-value industrial products synthesized by our bacterium, we are developing an optical biosensor on a paper-based support produced via wax printing. The sensor detects triacylglycerols using immobilized enzymes that generate a chromophore from the analyte. The color intensity correlates directly with the concentration of triacylglycerols in the sample. The detection is sensitive so it can detect even very low concentrations of the analyte of interest thanks to a cascade enzymatic reaction, and specific, so it can accurately identify and and respond only to the target analyte.