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
In the 21st century, the face of remediation is changing, and biotechnological solutions are increasingly replacing traditional chemical and physical methods, offering safer and more sustainable alternatives. Indeed, in bioremediation – the degradation of pollutants using living organisms – there are many bacterial strains reported to degrade pollutants and a vast body of literature describing them. However, what is often missing to make biology a true engineering discipline—one that is accurate, reproducible, and controlled—are a standardized methodology and a set of model organisms. This is where H.E.R.O. comes in. H.E.R.O. (High-performance Engineered Rhodococcus opacus) is a standardized bacterial chassis for bioremediation with two integrated components: C.A.P.E. (Computational Assistant for Pathway Engineering), a computational tool, and H.E.L.M.E.T. (High-performance Engineered Lipase-based Monitoring for Endogenous TAGs), a biosensor. Together, these are designed for applications in the synthetic biology of Rhodococcus opacus PD630. The system enables the rapid development of engineered strains capable of degrading pollutants while simultaneously producing high-value biomolecules.
History
Hi, there we are here to tell you a special story, the story of how H.E.R.O. was born...
Well, it all started in Bologna, where we were trying to tackle the growing problem of pollution in our oceans: POI FOTO
While digging through mountains of scientific literature for a solution, we stumbled upon a paper from Japan. It described a unique bacterium, Rhodococcus oxybenzonivorans, with an incredible ability: it could essentially eat BP3, breaking it down into harmless compounds. POI FOTO
At first, we were ecstatic. "This is it! The perfect answer!" But we hit a wall almost immediately. How were we supposed to work with a bacterium that was not only thousands of miles away, but was also a complete mystery to science? It was almost unusable for us.
Then, sitting around and brainstorming, an idea sparked: "What if we don't need the entire bacterium? What if we could just borrow its superpower?"
Our plan became clear: we would identify the specific genes responsible for degrading BP3 and transfer them into a reliable, well-understood bacterium that we use in the lab every day.
But that brilliant idea just opened a floodgate of new questions for us. Which bacterium would be the best new host? And more importantly, out of all the genes in the predicted cluster, which ones were the essential keys to the whole process?
That was our turning point. We realized we needed to think bigger. We needed more than just a single solution for a single problem. What we really needed were better tools for the job. We needed:
- A highly customizable bacterial platform that could be easily engineered for new tasks.
- A powerful predictive tool that could analyze gene clusters and tell us which parts were important.
Introduction - Why Rhodococcus?
The genus Rhodococcus represents a phylogenetically and catabolically diverse group of Gram-positive, non-motile, aerobic bacteria (Bell et al, 1998).
The peculiarities of the members of this genus are their ability to utilize a wide variety of carbon and energy sources (Ludwig et al., 2012) and their extreme and contaminated isolation environments (Kim et al., 2021; Xiang et al., 2022). These two main characteristics are highly connected to one another: their wide distribution is due to their extraordinary metabolic versatility, in addition to their unique environmental persistence and robustness (LeBlanc et al. 2008; Cappelletti et al. 2016).
Down here are five examples of Rhodococcus strains, click on them to reveal which pollutants they degrade! CARTE - Rhodococcus sp. 14 – PAHs (Song et al. 2011) - Rhodococcus ruber IEGM 231 – crude oil (Ivshina et al. 2016) - Rhodococcus rhodochrous BX2 – alinfatic nitriles (Fang et al. 2015) - Rhodococcus opacus 3D – Phenol (Anokhina et al. 2025) - Rhodococcus pyridinivorans P23 – PET (Guo et al. 2023)
But, what’s the Missing Piece?
Given the incredible potential of this genus, we must also recognize the challenges ahead. Many Rhodococcus strains have been identified as efficient pollutant degraders, but only a few are currently used in industry due to the limited availability of reliable genetic parts and engineering tools (de Lorenzo et al., 2018). To cover this "lab-to-market gap," it is important to develop a strain that meets strict criteria of economic feasibility, predictability, robustness, engineering flexibility, and safety. This is a demanding task, and many promising strains fail to pass such rigorous quality checks.
Therefore, we, the iGEM team from Bologna, are proposing a new chassis for bioremediation based on the strain Rhodococcus opacus PD630. This would be a customized platform for inserting useful genes from other degrader bacteria within the Rhodococcus genus. Such an approach promises to be cheaper and less wasteful.
CARTA RHODO We chose Rhodococcus opacus PD630 because it is a metabolically robust, genetically accessible bacterium (Firrincieli et al. 2022) with the ability to accumulate oily lipids called triacylglycerols (TAGs) up to 80% of its dry cell weight under the right conditions (Voss et al., 2001; Antony et al. 2024). Taken together, the combination of high lipid accumulation, broad catabolic flexibility, and a growing genetic toolkit firmly establishes Rhodococcus opacus PD630 as a versatile and valuable chassis for biotechnological applications, creating a cycle where pollutants are degraded and high-value compounds are produced.
Project outline – H.E.R.O.
H.E.R.O. aims to standardize genomic engineering in Rhodococcus opacus PD630 by creating a stable and characterized strain that can efficiently integrate new genes. To achieve this, we are integrating the gene for Cre recombinase (Kitagawa et al. 2023), a site-specific enzyme (Hamilton and Abremski, 1984), directly into the Rhodococcus opacus genome.
The Cre recombinase gene, paired with a loxLE site, will be placed under the control of an inducible pTip promoter (Nakashima et al. 2004). This ensures that the recombinase is only produced when needed, preventing constant and potentially harmful enzymatic activity. The entire cassette will be inserted into a previously identified "safe-harbor" site—a non-regulatory genomic region described by Antony et al. (2019)—to minimize disruption to the cell's natural functions
Fig: The genome of Rhodococcus opacus PD630 that has become HERO after the insertion of Cre/lox system. TipAL is the transcriptional regulator protein that controls the pTipA promoter. ThioR is the gene for the resistance to Thiostreptone.
So that is how it works:
To establish Rhodococcus opacus as an effective chassis organism for bioremediation, a well-characterized set of promoters is essential for achieving predictable and controllable gene expression. Therefore, our team is testing a toolkit of four different promoters. This set includes the common pLac promoter and three variants based on a native promoter from Rhodococcus jostii RHA1, which was previously studied by Round et al. (2019).
The key benefits are:
- Stable chromosomal integration Unlike plasmid-based expression, which requires constant antibiotic selection to be maintained, this system integrates genes directly into the bacterial chromosome. This ensures that the new genetic circuit is stably maintained and faithfully inherited by daughter cells, leading to more reliable and consistent strain performance.
- Elimination of antibiotic resistance plasmids After the gene of interest is integrated, no origin of replication is available in Rhodococcus. The final engineered strain therefore presents antibiotic-resistance genes only inside the genome and not on a plasmid, which minimizes the risk of spreading resistance genes via horizontal gene transfer. Furthermore, selective pressure is not required, allowing application in more environments.
- Standardized and Efficient Workflow
This system creates a "plug-and-play" platform for engineering Rhodococcus opacus. Once the master strain containing the inducible Cre recombinase is built, inserting any new gene becomes a routine process:
- Clone the gene of interest into the standardized delivery plasmid.
- Introduce the plasmid into the master strain.
- Induce Cre expression.
- Select recombinant strains. This standardized workflow increases the speed, efficiency, and reproducibility of genomic modifications.
- Precise and Predictable Integration The Cre-lox system is site-specific, meaning the new gene is inserted into a single, predetermined location in the genome. This avoids the risks associated with random integration, such as disrupting essential native genes or causing unpredictable expression due to "position effects." This precision leads to highly predictable outcomes and functionally consistent clones.
- High Versatility The master strain is a versatile chassis that can be used to integrate a wide variety of genetic payloads. Whether you're inserting a single gene, a complex metabolic pathway, or a sensor circuit, the core integration method remains the same. This makes the platform highly adaptable for future projects and diverse applications.
- Genetic toolkit
H.E.R.O. component: C.A.P.E.
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.
H.E.R.O. support: H.E.L.M.E.T
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.