Context
Since the successful Mars flyby by NASA’s Mariner 4 in 1965 , the red planet has become a primary focus of interplanetary exploration for NASA and other space enterprises worldwide. Over the past 50 years, numerous missions have been launched to study Martian geology, climate, and potential for supporting life- all with the ultimate objective of enabling human exploration. Despite recent advancements made by countless space agencies to achieve this, less than 50% of Mars missions have been successful . There are still many challenges that forestall the possibility of sustained human presence on Mars.
The Payload Problem
One of the biggest issues concerning Mars expeditions are the high expenses involved in transporting essential resources between Earth and Mars, also known as the Payload Cost . Transporting even the cheapest resources from Earth – such as basic plastics – can cost billions of dollars for a single round trip . In the early stages of human exploration, even simple polymers could become rare, high-value materials.
Launch of a Falcon 9 rocket
The Rise of Bioplastics
Back on Earth, efforts are underway to decrease the use of petroleum-based plastics in favor of carbon-negative bioplastics ; which are often derived from plant-based materials to form polymers such as Polylactic Acid (PLA), Polyethylene (PE), and variations of other plastics that are renewable and biodegradable . Another source of bioplastics comes from microorganisms which produce polyhydroxyalkanoates (PHA) (Chen et al., 2018) . These bioplastics have properties comparable to conventional plastics, but with the added benefits of being produced from sustainable resources.
PHA plastic pellets and structure
In other words, relying on E. coli as a source of on-site plastic production will save space companies millions of dollars from having to transport plastics or other resources from Earth all the way to Mars as well as reducing quantities of non-biodegradable plastics being produced on Earth.
What is PHBV?
PHBV is a PHA copolymer of polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV). By tuning the PHB:PHV ratio, the copolymer’s tensile strength, modulus, ductility, and thermal stability can be modulated to suit different applications.
PHB and PHV monomers make up the PHBV structure
As a result, this makes it a promising candidate to utilize in environments where resources are scarce. A leading organism in bioplastic production is Cupriavidus necator – a naturally autotrophic bacterium capable of producing PHB from carbon dioxide, making it a prime candidate for eco-friendly plastic production on Earth and potentially on Mars.
Why Not Use C. necator to Make Bioplastics?
C. necator is notoriously difficult to engineer due to slow genetic manipulation and limited resource availability. Meanwhile, E. coli is much easier to genetically alter. Scientists have seen success engineering E. coli to be autotrophic while others were able to engineer the bacteria to produce PHBV using acetate as a main carbon source (Gleizer et al., 2019; Nissan et al. 2024). So far, no system has combined both capabilities within a single framework: an autotrophic E. coli exporting acetate that a second E. coli converts to PHBV. None have integrated them as a CO₂-to-bioplastic co-culture.
Gleizer et al., 2019; Nissan et al. 2024
Using E. coli to Produce Bioplastics on Mars
The 2025 University of Rochester iGEM team aims to engineer a paired E. coli bioreactor system with the following two strains:
- Strain 1: Autotrophic and secretes acetate
- Strain 2: Grown on acetate secreted by Strain 1 and synthesizes PHBV under inducible control
As a proof of concept, our design draws inspiration from (Cestellos-Blanco et al.), who demonstrated a two-module process in which CO₂ is first biologically converted to acetate by the autotrophic acetogen Sporomusa ovata , then used directly – without media purification – by Cupriavidus basilensis for PHB production.
Cestellos-Blanco et al.
Adapting this motif to paired E. coli chassis gives us a practical platform for rapid iteration in a well-characterized organism: both strains share the same media, parts, and regulatory logic, which reduces interspecies integration costs and enables tighter, chassis-wide control. Additionally, we add a bicarbonate-sensing control layer. When intracellular HCO₃⁻ rises, soluble adenylyl cyclase (sAC) produces cAMP; cAMP activates the cAMP receptor protein (CRP), which enhances transcription from CRP-controlled promoters , leading to upregulation of target genes as inorganic carbon (CO₂/HCO₃⁻) increases.
On top of serving as an environmentally friendly alternative to current plastics, our approach is designed to operate in environments with high CO₂ levels, such as the Martian atmosphere, which is ~95% CO₂, so that carbon fixed by the autotrophic strain can be routed to PHBV production by its partner. Combined with 3D printing systems, the copolymer can be fabricated on-site into essential resources including spare parts, basic medical equipment, storage containers for food and water, fibers, and potentially minor infrastructure.
Moreover, excess acetate can be incorporated into biofertilizers to enhance soil productivity in extraterrestrial environments. Engineering E. coli for In-Situ Resource Utilization (ISRU) to produce PHBV and acetate provides a foundation to produce materials on-site instead of relying on return trips to Earth – which can help significantly reduce payload costs while supporting Martian agricultural efforts, bringing us one step closer to human exploration on Mars.
Learn About Our Project
- 1. Making Autotrophic E. coli
- Discover how we engineer E. coli to utilize carbon dioxide from Mars’ atmosphere as a carbon source
- 2. Producing Acetate
- Explore our acetate module, which directs metabolism toward acetate output for capture, cross-feeding, or other external uses
- 3. Citrate Synthase Knockdown via CRISPRi
- Learn about how we downregulate citrate synthase to enhance PHBV and acetate production
- 4. CO 2 -Responsive PHBV
- Discover our CO₂-responsive PHBV module that turns cross-fed acetate from a separate E. coli into bioplastic.
Metabolic map of our project modules
Strain 1: Emulating Acetogenesis - From CO 2 to Acetate
Genes in each plasmid for Strain 1
We propose engineering an E. coli BL21 (DE3) strain for autotrophic growth by expressing key Calvin-cycle components (Nissan et al., 2024). Form I RuBisCO from Rhodobacter capsulatus catalyzes RuBP carboxylation (RuBP + CO₂ + H₂O → 2 × 3-phosphoglycerate). The RuBisCO chaperone RbcX from Synechococcus sp. PCC 7002 assists folding and assembly – stabilizing CbbL C-termini and promoting formation of the L₈ core prior to rbcS addition to yield the L₈S₈ Form I RuBisCO holoenzyme. Phosphoribulokinase (prkA) from Rhodobacter sphaeroides regenerates RuBP (ribulose-5-phosphate + ATP → RuBP + ADP).
rbcX, prkA, and ruBisCO pathway
To supply reducing power, we express NAD⁺-dependent formate dehydrogenase (FDH) from Pseudomonas sp. 101 (HCOO⁻ + NAD⁺ → CO₂ + NADH + H⁺), and to increase local CO₂ availability at RuBisCO, we express β-carbonic anhydrase from Dolichospermum circinale (CO₂ + H₂O ⇌ HCO₃⁻ + H⁺), which accelerates CO₂/HCO₃⁻ interconversion and helps maintain conditions favoring RuBisCO carboxylase activity.
Beta Carbonic Anhydrase and NAD+ Dependent Formate Dehydrogenase
Our overarching goal is an E. coli strain that fixes atmospheric CO₂ as its primary carbon source, reducing dependence on imported sugars and enabling more sustainable biomanufacturing in resource-limited settings.
To drive acetate overproduction , we will additionally express the pta–ackA pathway under the control of a rhamnose-inducible promote r. Phosphate acetyltransferase (pta) from Clostridium kluyveri catalyzes the conversion of acetyl-CoA and inorganic phosphate into acetyl-phosphate , while acetate kinase (ackA) from Bacillus subtilis converts acetyl-phosphate and ADP into acetate and ATP ( Létisse, F. 2017) . Together these enzymes channel acetyl-CoA into the acetate overflow pathway, driving its conversion to secreted acetate from an inorganic carbon source.
Conceptually, this strain mirrors the terminal steps of acetogenesis. Whereas acetogenic bacteria, such as Sporomusa ovata , reduces CO₂ to acetyl-CoA via the Wood–Ljungdahl pathway and then converts acetyl-CoA to acetate through Pta–AckA (Ragsdale, S. W., & Pierce, E. 2008), our design fixes CO₂ via the Calvin (RuBisCO/PRK) cycle, routes carbon to acetyl-CoA in central metabolism, and employs the same Pta–AckA node to secrete acetate. In this sense we generate an acetate-producing autotrophic phenotype without the Wood–Ljungdahl pathway – trading Wood–Ljungdahl pathway’s enzyme complexity and oxygen sensitivity for a modular, well-characterized E. coli chassis.
Strain 2: PHBV Production from Acetate
Genes in each plasmid for Strain 2
A parallel strain will be transformed with a modified phaBCA operon assembled with different recombinant genes – phaC from Allochromatium vinosum , phaA replaced by bktB from Haloferax mediterranei (Hou et al., 2013), and phaB from Synechocystis sp. PCC 6803. Altogether, this operon is responsible for the production of PHB/PHBV based on available substrates. The H. mediterranei β-ketothiolase BktB condenses acetyl-CoA with either another acetyl-CoA (to yield acetoacetyl-CoA, precursor for PHB) or propionyl-CoA (to yield 3-ketovaleryl-CoA, precursor for PHV). PhaB reduces acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA and 3-ketovaleryl-CoA to (R)-3-hydroxyvaleryl-CoA, and PhaC polymerizes hydroxyacyl-CoA monomers into PHB or PHBV, with the final composition determined by the balance of acetyl-CoA versus propionyl-CoA precursors .
The operon will be combined alongside tdcB and tdcE , which function together to provide propionyl-CoA: TdcB (L-threonine dehydratase) converts L-threonine to 2-ketobutyrate, and TdcE (glycyl-radical enzyme) cleaves 2-ketobutyrate to propionyl-CoA + formate. The overall route channels threonine into propionyl-CoA, which is the essential C3 unit needed for incorporation of 3-hydroxyvalerate (3HV) into PHBV ( Hou et al., 2017) . Incorporating propionyl-CoA expands the polymer from pure PHB into the copolymer PHBV, improving its material properties (greater ductility, toughness, and thermal stability compared to brittle PHB).
TdcE and TdcB pathway
In this PHBV strain, we will also tune expression of the thioesterases TesB and YciA: calibrated thioesterases that hydrolyze excess acyl-CoAs to free acids, regenerate CoA-SH, and relieve acyl-CoA crowding, which in practice improves assimilation of imported acetate into acetyl-CoA and supports downstream PHBV synthesis – consistent with TesB/YciA-mediated gains in 3-HB flux reported in cyanobacteria and E. coli. ( Guevara et. al 2019; Ku, J. T., & Lan, E. I. 2018).
Thioesterase pathway
Bioplastic Production in High CO 2 Conditions
Our tunable CO₂-responsive system leverages components already present in the autotrophic E. coli chassis . Under elevated CO 2 conditions , α-carbonic anhydrase (an enzyme that converts CO₂ into bicarbonate) will be expressed to support autotrophic growth . This bicarbonate activates a truncated version of human soluble adenylyl cyclase (sAC) , leading to elevated intracellular cAMP . The cAMP then binds to a cAMP receptor protein (CRP) , which activates transcription from a CRP-regulated promoter placed upstream of the phaBC(bktB) and tdcB-tdcE operons. This architecture ensures that PHBV biosynthesis is only induced when CO₂ levels are relatively high .
Promoter pathway
Promoters driving phaBC(bktB) and tdcB/tdcE carry an upstream CRP box; cAMP-bound CRP binds this site and activates transcription, so gene expression increases in response to higher intracellular HCO₃⁻ (from CO₂).
Applications of Acetate Production
Beyond serving as a cross-feed for our PHBV-producing strain, acetate is a versatile product with stand-alone applications. On Mars , our system will complement our sustainable efforts by producing acetate, serving as a counter-ion that can be combined with other plant nutrients such as nickel, calcium, zinc to enhance soil productivity in the native Martian regolith. We also plan to evaluate the performance of the CO₂-responsive promoter under simulated Martian atmospheric conditions (~95% CO₂) to assess its induction efficiency . Incorporating experiments in a microgravity simulator (such as our clinostat Spinova) will further validate the system’s relevance for space-based applications.
Acetate as a potential biofertilizer for plants
Citrate Synthase Knockdown via CRISPRi
In our PHBV producing strain, we will implement CRISPRi-mediated knockdown of citrate synthase ( Heo, et al., 2017; Larson et al., 2013; Qi et al., 2013) in a separate strain as a proof of concept for pathway-level modulation . Studies have shown that reducing citrate synthase activity increases intracellular acetyl-CoA availability, leading to enhanced PHBV production . This modular approach demonstrates the flexibility of our chassis design and paves the way for future integration of dynamic regulatory systems. The CRISPRi module involves introducing recombinant sadCas9 from Staphylococcus aureus – selected for its smaller footprint and 5′-NNGRRT-3′ PAM, which allow closer transcription start site targeting and typically stronger CRISPRi than NGG-limited Sp-dCas9 – together with an sgRNA that targets an inserted recognition sequence (gltArec) upstream of the endogenous E. coli citrate synthase gene (gltA) for transcriptional knockdown.
CRISPRi
The citric acid cycle represents a competing flux for acetyl-CoA, which can otherwise be directed toward acetate or PHB(V) synthesis . Previous studies have shown that gltA knockdown can enhance PHB production by increasing acetyl-CoA availability ( Park et al., 1994; Rajpurohit & Eiteman, 2024) .
Summary
PHAntom is more than just a biomanufacturing platform ; it represents a vision for sustainable space colonization. By merging cutting-edge advances in synthetic biology with the constraints and opportunities of Mars , we offer a scalable, modular solution to create materials from microbes and the Martian atmosphere . PHAntom turns the dream of off-world living into a biological reality .
