Entrepreneurship

1. The problem

The logistics of producing and storing perishable foods, especially fruits and vegetables, remains a major global challenge. According to FAO (United Nations Food and Agriculture Organization), around one-third of global fruit and vegetable production is lost across the supply chain, from harvest to transportation and storage.

The citrus market is no exception and is also highly vulnerable to diseases that severely impact production. Worldwide, the most destructive challenge in citrus farms is Greening, caused by the bacterium Candidatus Liberibacter spp. Globally, Greening has already led to the destruction of more than 60 million citrus trees, collapsing production in entire regions such as India, Indonesia, Africa, and Florida (USA). In Brazil, the world’s largest citrus producer, the fruit drop rate caused solely by Greening in the 2024/25 harvest was about 25%. To make matters worse, in the absence of effective control for this bacterium, current management relies on the intensive use of insecticides, and eradication of infected trees. However, these approaches face critical limitations: only partial effectiveness, environmental risks, stricter regulatory restrictions, and escalating production costs that undermine profitability.

Post-harvest challenges are equally significant, with pathogens that directly damage stored oranges. The fungus Penicillium digitatum, responsible for Green mold, accounts for up to 90% of post-harvest losses, rapidly deteriorating oranges and drastically reducing shelf life. Also, Sour rot (caused by Geotrichum candidum) is responsible for substantial losses, especially in high-rainfall seasons. Together, these diseases cause nearly 50% of losses of all oranges produced, compromising fruit availability in a short time.

Given this scenario, it becomes clear that current control methods suffer from a lack of effectiveness, failing to access the problem and provide a sustainable or long-term response. Consequently, the sector urgently requires innovative alternatives that combine pathogen control efficacy with environmental safety, regulatory compliance, and economic feasibility. This gap creates a structural global challenge that impacts not only stakeholders such as farmers and industries but also consumers and entire communities dependent on this economy.

2. Opportunity

Reduced supply raises production costs, undermines fruit quality, and increases prices of citrus derivatives such as juices and essential oils. In a sector worth billions of dollars, reducing losses not only improves profit margins but also strengthens international competitiveness.

To put things into perspective, region has 362,160 hectares of citrus under cultivation, with about 182.71 million trees currently in production. According to stakeholder insights, virtually the entire area is already contaminated with Greening. After evaluating different aspects of how the disease works with a cellular automaton-based computational model, the estimated demand per harvest would be around 15 mg per tree. This translates into approximately 3 tons of peptide required annually for Greening treatment, and the market opportunity for post-harvest diseases (Green mold and Sour Rot) would be even greater. If we consider the application of 5 mg of peptide per kg of oranges, equivalent to the maximum dose of the fungicide imazalil currently permitted in Brazil, the market demand would reach around 20 tons annually of CTX annually, just to cover the 40 million tons of fruits destined for fresh consumption.

Additionally, in the absence of effective control for these diseases, the current management relies on the intensive use of insecticides and fungicides. According to the Food and Agriculture Organization of the United Nations (FAO), global agriculture used 3.5 million tons of agrochemicals in 2021, twice the volume reported in 1991, demonstrating a steady upward trend in pesticide consumption. In the same year, Brazil applied 719,500 tons, ranking as the world’s largest consumer of pesticides (topic further discussed on our Citrus Diseases component). Especially in citrus production, according to the Pesticide Action Network (PAN), oranges are among the 12 most contaminated foods with agrochemicals, with substances classified as Highly Hazardous Pesticides (HHPs) by the UN. From an environmental perspective, the lack of specificity of pesticides leads to the intoxication of non-target organisms. As a result, the biological processes of these living beings are compromised, disrupting the ecological balance. Also, the indiscriminate use of antibiotics against Greening raises concerns about the potential emergence of resistant strains.

Against this backdrop, the market opportunity is clear: to develop innovative solutions capable of reducing citrus losses, extending fruit shelf life, and ensuring large-scale production, while also meeting sustainability and regulatory requirements. In an expanding sector with limited effective tools, the stage is set for disruptive biotechnological solutions.

3. Our solution

Our solution is grounded in a synthetic biology strategy for the large-scale production of AMP, specifically CTX, aimed at controlling the major pests and pathogens that threaten the citrus chain, including Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum). Unlike conventional chemical pesticides, our biotechnology-based solution leverages sustainable and safe mechanisms to effectively suppress the diseases through killing and inhibiting the growth of fungus and bacteria. We designed a production process grounded in circular economy principles, transforming useless orange peel residues processed from the juice industry to valuable feedstock for CTX biosynthesis. This positions our technology as a high-impact alternative designed to address the urgent need for innovation in the citrus industry, bridging cutting-edge science with tangible economic and social impact.

4. What, How, Why

We target the major phytopathologies of the citrus chain - Greening, Green Mold, and Sour Rot - by applying CTX, a powerful AMP. To make this possible, our team developed three biotechnological platforms for CTX production: Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae. Our ultimate goal is to protect oranges and orchards, ensuring a stable supply of citrus for both local and global markets. For clarity, we structured our strategy around three guiding questions:

What? Apply CTX, a powerful AMP, for controlling pests and pathogens in citrus crops, targeting phytopathologies such as Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum).

How? Leveraging the principles of synthetic biology, our focus is to produce CTX using genetically engineered Escherichia coli, Saccharomyces cerevisiae and Aspergillus oryzae capable of converting carbon sources into AMPs with high efficiency, low cost, and scalable production.

Why? To apply synthetic biology to safeguard citrus production by providing biotechnological solutions that increase yield, improve the livelihoods of growers, and ensure the availability of oranges in local and global markets.

A clear business description is essential to communicate the problem being solved, the innovation offered, and the market opportunity it addresses. Our business addresses one of the most pressing challenges in global agriculture: the high vulnerability of citrus crops to pre- and post-harvest diseases. With Greening, Sour Rot, and Green Mold, growers urgently need effective and sustainable alternatives to costly and environmentally damaging chemical inputs. Our innovation leverages Synthetic Biology to pioneer the first application of the antimicrobial peptide CTX against key citrus pathogens (Candidatus Liberibacter spp. causing Greening, Penicillium digitatum causing Green Mold, and Geotrichum candidum causing Sour Rot), offering efficacy at low concentrations. By integrating circular economy practices, such as the use of citrus industry by-products as fermentation substrates, and deploying scalable microbial biofactories based on Aspergillus oryzae, we provide a cost-effective, environmentally responsible, and market-differentiated solution. Positioned within the expanding global biodefensives sector, our technology not only protects citrus production but also enhances grower profitability, strengthens food security, and meets rising consumer and regulatory demand for sustainable agriculture.

1. Mission, Vision and Values

In building our venture, we recognize that technological innovation alone is not enough – it must be guided by a clear sense of purpose and responsibility. Our mission defines what we seek to achieve today, our vision reflects the long-term impact we aspire to create, and our values establish the principles that guide every decision and action we take. Together, they form the foundation of our commitment to transforming citrus production through biotechnology in a way that is sustainable, inclusive, and scientifically rigorous.

Mission: To combat phytopathologies such as Greening, Green mold, and Sour rot through the application of antimicrobial peptides (AMPs), establishing technological production platforms to safeguard citrus farming. By doing so, we aim to reduce pre- and post-harvest losses, thereby increasing productivity and ensuring greater availability of oranges in the market.

Vision: To become a global leader in the development and application of AMPs for sustainable citrus production.

Values:

1. Driving innovation in the biotechnological development of production platforms
2. Implementing sustainable systems grounded in circular economy principles
3. Integrating stakeholder perspectives into technological and scientific advancement
4. Upholding responsibility and integrity in science

2. Business model canvas

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The business model was designed with a clear focus on the production, application, and market positioning of the peptide as the project's core activity. Its development took place in a collaborative workshop that brought together representatives from all key areas of our team. Through structured discussions and active participation, each component of the canvas was carefully proposed, debated, and refined. After thorough analysis and integration of all contributions, we consolidated a cohesive version of the business model that reflects our collective vision and strategic alignment.

The purpose of this business model is to clearly articulate the project's value proposition and define its key market segments from an entrepreneurial and innovation-driven perspective. It provides a strategic framework to guide decisions on product development, value chain integration, and go-to-market strategies.

In the context of synthetic biology and agricultural biotechnology, the model strengthens our understanding of the technological, regulatory, and economic factors that shape the viability of our peptide-based solution. It also plays a central role in potential sponsors, enabling effective communication with potential partners, incubators, investors, and public institutions.

By clearly outlining our positioning and potential impact, this business model has facilitated the formation of meaningful partnerships and articulated a coherent path from early-stage research to real-world application and scalable implementation.

We aim to deliver a biotechnological solution for the control of phytopathogens in citrus crops. Through a synthetic biology platform optimized for the production of AMPs, we seek to establish a circular economy model that provides an innovative and environmentally responsible product. Our solution proposes an innovative approach to combat phytopathologies such as Greening, Green Mold, and Sour Rot through the use of AMPs. By establishing a biotechnological production platform, therefore boosting productivity, and ensurind a steady supply of oranges to the market.

Customer Segments

1. Orange producers, farmers, and juice industry
2. Agricultural defensives formulators

Our target market spans the entire citrus value chain, from crop to food processing and storage. This includes citrus growers of all scales and production purposes, from smallholder farmers to large-scale exporters. In addition, we target agribusiness companies focused on biotechnology-based formulations, agricultural inputs, and crop and storage protection, as well as agrochemical formulators and biotech developers seeking to integrate peptide-based bioactives into their portfolios. Finally, by supporting the orange juice industry and fresh fruit consumption markets, both of which depend on consistent fruit quality and availability, our solution contributes directly to sustaining consumer demand and global trade in citrus products.

Channels

1. Pepcitrus website
2. Marketplaces integrated with social media
3. Own commercial representatives
4. Agriculture formulation companies
5. Sector-specific events and symposia

Our solution can be distributed through a multichannel strategy, including the official Pepcitrus website and integrated social media marketplaces. Direct sales will be supported by our commercial representatives and strategic partnerships with companies in the agricultural inputs sector. We will also promote our innovation through participation in targeted agricultural events and scientific symposia.

Customer Relationships

We are committed to providing comprehensive support services, including post-sale assessing and evaluating peptide efficacy in the field. To enhance this process, our hardware-based detection system enables precise, real-time monitoring of peptide activity directly at the application site, ensuring accurate performance tracking and early identification of effectiveness patterns. In addition, our product will be associated with certifications and sustainability seals that emphasize circular economy practices, national origin, organic compatibility, and the use of sustainable biotechnological inputs, thereby adding differentiated market value for our customers.

Revenue Streams

1. Sales of peptides focused on pre- and post-harvest control of diseases such as Greening, green mold and sour rot
2. Development of customized solutions for other pathogens through our in-house optimized pipeline
3. Technology transfer and licensing of technologies directly and indirectly related to the main product
4. Sales to the government

Our primary source of revenue will be the sale of peptide-based solutions for pre-and post-harvest management of citrus diseases, such as Green Mold, Sour Rot, and Greening. Future revenue streams will include the development of customized biotechnological solutions for other pathogens, alongside technology transfer and licensing of both core and derivative innovations. We will also pursue sales through government contracts and public sector procurement programs, expanding adoption and ensuring long-term scalability.

Key Resources

1. Intellectual capital for scaling up through biotechnology
2. Material resources from Unicamp (currently and in the future as an incubated company)
3. Qualified and motivated team
4. Sponsors and partner companies
5. Public/private laboratories (through partnerships or rental)
6. Production plant and inputs
7. Residues for production

Our current key resources include specialized high-level intellectual capital focused on scaling synthetic biology applications, access to advanced research infrastructure at Unicamp (with potential incubation opportunities), and a highly qualified and motivated team. Looking ahead, we will leverage strategic partnerships with private and public laboratories, crowdfunding initiatives, industry sponsorships, and access to productive plants and citrus waste streams as raw materials for production.

Key Activities

1. Development of technological innovation through synthetic biology
2. Peptide production through sustainable routes
3. Testing of different chassis for efficient peptide production
4. Development of solutions that are environmentally sustainable, simpler, and lower in cost

Our operations are centered on the development of innovative technologies through synthetic biology. We focus on environmentally sustainable peptide production pathways and the optimization of microbial chassis for high-efficiency peptide synthesis. We are also dedicated to develop accessible, cost-effective solutions with low environmental impact, ensuring scalability and market feasibility.

Key Partnerships

1. Small and medium-sized orange producers
2. Science and technology institutions – Unicamp, IAC
3. Citrus-related companies that can provide residues
4. Citrus growers' cooperatives
5. CROs
6. AlfaCitrus

Our strategic partnerships include small and medium-sized citrus producers, scientific and technological institutions such as Unicamp and IAC, citrus cooperatives, CROs (Contract Research Organizations), and large-scale producing companies like Alfa-Citrus that can supply raw materials such as agro-industrial residues. These collaborations are essential for raw material supply, co-development, and pilot testing.

Cost Structure

1. Production (equipment, reagents, inputs, bioreactors, consumables)
2. Specialized personnel
3. R&D (Research and Development)
4. Marketing and outreach
5. Logistics and distribution
6. Product regulation
7. Consultants and legal (marketing, finance, legal advisors)

Our main costs include production (equipment, reagents, bioreactors, and consumables), specialized personnel, research and development, marketing and outreach, logistics and product distribution, regulatory compliance, and external consultancy (marketing, finance, and legal services). These components are essential for ensuring product quality, scalability, and market competitiveness.

3. SWOT analysis

The SWOT analysis provides a comprehensive overview of the internal and external factors that shape the strategic positioning of Pepcitrus, our biotechnology-based initiative to protect citrus crops through the development and application of AMPs. By mapping out the project's strengths, weaknesses, opportunities, and threats, we can better understand the potential for innovation, scalability, and impact in the citrus sector. This analysis highlights the competitive advantages derived from scientific expertise and technological innovation, identifies the challenges that may limit short-term scalability, and outlines the market opportunities and external risks that influence the success of Pepcitrus.

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Our venture benefits from strong institutional support from Unicamp, access to advanced research infrastructure, and a highly qualified biotechnology team, enabling innovation in sustainable citrus disease management. Key strengths include stakeholder relationships, technological capacity, and international visibility through iGEM. While challenges such as limited financial resources, dependence on imported inputs, and laboratory-scale production exist, strategic partnerships and scale-up planning can mitigate these risks. The Brazilian citrus market offers significant opportunities due to its size, the high-impact nature of diseases like Green Mold, Greening, and Sour Rot, and the lack of low-environmental-impact biotechnological solutions. Threats, including low-cost chemical alternatives, regulatory delays, and resistance among traditional growers, can be addressed through value differentiation and stakeholder engagement.

Overall, the SWOT analysis confirms that our company is strategically positioned to capitalize on market gaps while building long-term competitive advantages through sustainable innovation.

In this context, the capacity for peptide production has been demonstrated across different microorganisms (Escherichia coli, Saccharomyces cerevisiae and Aspergillus oryzae) and production systems (liquid fermentation and solid fermentation). Among the four models, the Aspergillus oryzae cultivated in a solid-state fermentation model stands out as the most promising for industrial applications, combining high titer, high production rate, and the ability to use residues as feedstock, reducing costs and increasing sustainability. Therefore, these findings highlight the robustness and scalability of Aspergillus oryzae when cultivated in a solid-state fermentation model.

Our TRY analysis evaluated different biofactorys (Escherichia coli, Saccharomyces cerevisiae and Aspergillus oryzae) in various growth conditions: liquid state fermentation and solid state fermentation. Among the four models, the Aspergillus oryzae cultivated in a solid-state fermentation model stands out as the most promising for industrial applications, combining high titer, high production rate, and the ability to use residues as feedstock, reducing costs and increasing sustainability.

Citrus cultivation is a cornerstone of the Brazilian economy, particularly in the Citrus Belt, formed by states of São Paulo and Southwest Minas Gerais, responsible for 80% of the country's production. Brazil is also the world's largest producer and exporter of orange juice, representing approximately 70% of global production and 75% of international trade.

However, the sector competitive advantagesfaces significant challenges, primarily due to diseases such as Greening. This incurable disease has caused severe productivity losses across orchards. In the 2023/24 harvest, farmers in the Citrus Belt produced 307.22 million boxes of oranges equivalent to a loss of 76,35 million boxes. However, due to the combined effects of diseases such as greening and adverse climatic conditions, production for 2024/25 was projected to drop sharply to 230.87 million boxes- a 24.85% decline. Together with climate change, these diseases pose significant threats to citrus productivity and sustainability, generating economic and social losses within the Brazilian citrus supply chain and impacting the global distribution of fresh and processed citrus products.

Beyond cultivation, the post-harvest phase is critical for ensuring fruit quality and shelf life. Key post-harvest diseases include green mold, caused by Penicillium digitatum, and sour rot, caused by Geotrichum candidum, which can result in losses exceeding 50% due to poor storage and handling practices.

In this context, current solutions remain limited, costly, and largely ineffective. Specifically for greening, strategies range from extensive removal of affected orchards to the application of antibiotics directly into infected trees, which leads to productivity losses, antimicrobial resistance, and negative externalities affecting consumers. Therefore, innovative and sustainable solutions are essential to ensure the long-term viability of the citrus sector. With this in mind, our company aims to transform the landscape by leveraging biotechnology to develop a definitive, scalable, and environmentally low-impact solution.

a. Market Analysis

The global citrus industry ranks among the most significant agribusiness sectors worldwide, both in production volume and economic impact. According to the USDA Foreign Agriculture Service, global orange production for the 2024/25 season is projected to reach 45.22 million tons.

The citrus value chain moves billions of dollars in exports and generates millions of direct and indirect jobs worldwide, making it a strategic sector for multiple economies, especially for Brazil. Our country holds an undisputed leadership in the global citrus market, being the largest producer of orange juice (representing 70% of the global market), and orange fruits (accounting for 29% of the global market).

Brazil dominates the global orange juice trade with output expected to reach 1.01 million metric tons in the 2024/25 season, primarily supplying Europe, the United States, and Asia. In 2023, Brazilian citrus exports totaled USD 220 million, with key markets including the Netherlands, United Kingdom, Germany, Switzerland, and Spain. This strong reliance on the European market underscores the urgency of adopting sustainable, low-pesticide production strategies.

When it comes to fresh oranges, Brazil is also the leader in world production with production at 13 million metric tons. In January 2025 alone, Brazilian citrus exports reached USD 14.9 million, while imports totaled USD 11 million, yielding a positive trade balance of USD 3.92 million.

This dominance is not new, as the country's favorable climate, vast cultivated areas, and consolidated infrastructure for processing and export have long sustained its competitive edge. Historically, citrus farming in Brazil began in the 16th century, when Portuguese colonizers introduced sweet orange seeds to Bahia and São Paulo. Benefiting from the favorable climate conditions of the Southeast, production expanded rapidly, and by the late 20th century Brazil had established itself as the world's leading producer and exporter of both fresh oranges and concentrated orange juice.

The Brazilian citrus industry plays a vital economic role generating 200,000 direct and indirect jobs and contributing with 189 million dollars in tax revenue (2024). This directly impacts the lives of millions of people and represents a market of critical importance within Brazilian society.

Despite its global leadership, Brazil's citrus industry faces critical challenges. Historical data reveal a consistent decline in productivity: between 1994/95 and 2003/04, the average output was 351 million boxes (40.8kg); it dropped to 337 million in the following decade and 308 million between 2014/15 and 2023/24.

The 2024/25 harvest in the citrus belt reached only 230,87 million boxes — an expressive decrease of 76.35 million boxes (24,85%) compared to the previous season. This sharp reduction is mainly attributed to climate variability and the high incidence of greening disease, which alone caused an estimated loss of 32 million boxes in the previous harvest. The number of productive trees also declined by 4.4% compared to the prior cycle, while premature fruit drop remains high, projected at 18.5%.

The constrained supply has directly impacted prices. In 2024, the price per box of oranges reached BRL 85 (US$15.57*), the highest level since 1994. Between October 2023 and October 2024, prices more than doubled, rising from BRL 54.25 to BRL 125 – an increase of 130%. For consumers, the inflationary effect has been dramatic: in September 2024, orange prices were nine times higher than in the same month the previous year, reflecting a 65.9% variation in the FGV price index (national index provided by Fundação Getulio Vargas). Global factors such as juice stock levels, pest and disease outbreaks, and climate events have amplified this price volatility.

Given the strategic importance of citrus farming for both Brazil and the global market, the sector's future depends on more than just maintaining productivity, it requires a low cost and high efficiency sustainable product. Producers, cooperatives, juice processors, exporters, government agencies, and consumers all play a central role in shaping this transformation. Beyond its relevance to Brazil's trade balance, citrus cultivation supports regional economies, provides employment, and ensures access to food on a global scale.

To remain a global leader in this billion-dollar industry, Brazil must not only expand and modernize its citrus belt but also adopt sustainable solutions that mitigate the impacts of climate change and combat devastating diseases such as Greening. Emphasizing environmentally responsible practices will reduce dependence on harmful chemicals, enhance soil and ecosystem health, and align the industry with global sustainability goals.

Equally critical is ensuring that small and medium-scale farmers, especially vulnerable to productivity losses, benefit from accessible, affordable, and scalable technologies. By empowering these producers with innovative bio-based tools, the industry can promote inclusivity and strengthen local economies.

In this way, Pepcitrus is not only a path to maintaining Brazil's leadership in citrus production with sustainability but also an opportunity to foster social equity, environmental stewardship, and long-term competitiveness in global agricultural markets.

b. Customer needs

Customer Pain Points

1. Citrus Producers: Experience significant economic losses caused by diseases for which effective control methods remain limited
2. Agrochemical Formulators: Dependence on conventional fungicides exposes them to regulatory restrictions, market resistance, and the absence of sustainable, differentiated products.
3. Agribusiness Companies: Lack of bioactive molecules to expand their portfolios.
4. Biotech Developers: Need validated compounds with scalable, cost-effective production systems; current technologies fail to balance efficacy, affordability, and industrial readiness.
5. End Consumers / Food & Nutrition market: Rising consumer demand for pesticide-free fruits and sustainable production methods reflects growing concern for global food security.

Citrus producers, from smallholders to large exporters, struggle with high economic losses caused by persistent diseases. Current chemical solutions are either ineffective, unavailable for certain pathogens, or increasingly restricted by regulation. This creates a costly dependency on inputs that do not fully solve the problem, threatening both productivity and profitability. Beyond farm-level economics, these losses cascade into the food chain, limiting availability of fresh fruit and juice for consumers and raising concerns over food security.

Agribusiness companies and agrochemical formulators face a parallel challenge: the market demands innovation, but their portfolios remain heavily reliant on conventional pesticides. With growing environmental restrictions and consumer pressure for low-residue products, the lack of biotechnological solutions creates a gap that limits competitiveness. For companies, this is not only a technical bottleneck but also a reputational risk, as the global food and nutrition sector increasingly values transparency, sustainability, and health-driven innovation.

Biotechnology formulation companies and developers are equally pressed to secure novel bioactives with scalable production systems. Their main issue lies in accessing compounds that have proven efficacy, can be produced at industrial scale, and meet the economic feasibility required for commercialization. At the same time, the citrus industry sits at the intersection of agriculture and nutrition: it provides key sources of vitamin C and other nutrients worldwide. A technology that combines innovation, sustainability, and scalability not only solves the challenges faced by growers and companies but also guarantees consumers access to safe, high-quality citrus. Our CTX-based solution tackles all these challenges head-on, delivering a smarter, greener, and more effective approach to citrus protection.

a. Inventiveness and scalability

Inventiveness

1. Novel application of the antimicrobial peptide CTX, never tested before against major citrus pathogens (P. digitatum, G. candidum and Candidatus Liberibacter spp.).
2. Novel scale-up strategy for commercial peptide production.
3. Demonstrated efficacy at low concentrations relative to current products, including diseases with no current commercial treatment (e.g., Sour Rot).
4. Circular economy integration: valorization of orange juice by-products as fermentation substrates.
5. Differentiation from conventional fungicides and the absence of equivalent biotechnological solutions in the current market.
6. Implementation of smart detection hardware that provides on-site, real-time insights into peptide performance, enhancing user experience and fostering continuous technical engagement.

Scalability

1. Transition from proof-of-concept efficacy to industrial feasibility via microbial biofactories.
2. Use of Aspergillus oryzae as a high-capacity secretion platform for cost-effective peptide production.
3. Evaluation of citrus production chain residues as feedstocks.
4. Reduced dependency on chemical peptide synthesis, lowering production costs and environmental impact.
5. Enable the production of other AMPs from agro-industrial residues, lowering costs and opening opportunities for the development of new biotechnological applications.
6. Potential for expansion into the growing bioinputs market, with direct alignment to global sustainability demands.

Current solutions for citrus diseases rely almost exclusively on chemical fungicides and bactericides, which present critical limitations: pathogen resistance, environmental toxicity, and regulatory restrictions. Moreover, for key diseases such as Sour Rot, there is no effective commercial solution available, leaving producers exposed to substantial post-harvest losses. While antimicrobial peptides have been reported in academic literature, no prior research or market product has demonstrated their application against the major citrus pathogens Green Mold, Sour Rot, and Greening. The absence of biotechnological alternatives highlights a clear innovation gap in the sector.

Our solution advances beyond existing knowledge by introducing CTX, an AMP originally isolated from the Cerrado frog Hypsiboas albopunctatus, as a novel antifungal and antibacterial agent for citrus protection. Laboratory testing revealed CTX’s inhibitory activity at low concentrations against Penicillium digitatum (90% of post-harvest orange losses) and Geotrichum candidum (Sour Rot, with no available fungicide treatment). Additionally, collaborative efforts extended its evaluation to Candidatus Liberibacter spp., the causal agent of Greening. By targeting pre-and post-harvest pathogens with four biotechnologicals approaches, this project represents an inventive and non-obvious solution compared to conventional chemical and academic strategies. Furthermore, our detection hardware strengthens customer relationships by enabling real-time monitoring and data collection on peptide performance on the field. This technological capability allows clients to make data-driven decisions and optimize application strategie. By integrating the hardware into our service ecosystem, we ensure a continuous assesment on the effectiveness of our product, fostering trust, transparency, and long-term collaboration grounded in measurable results.

Our project has evaluated and compared different microbial factories concerning their impact on the putative production process, including: (i) the complexity of genetically engineering the host; (ii) the possibility of using citrus by-products as feedstocks; (iii) the impact on the bioprocess complexity, including the necessary unitary steps of a production plant, media requirements and downstream processing steps. Thus, we selected three different hosts - Escherichia coli, Saccharomyces cerevisiae, and the Aspergillus oryzae - to evaluate their production capacity.

Therefore, we have compared our candidates for microbial factories and chosen because of the evaluation below:

1. Escherichia coli is a widely used host for recombinant protein production due to its advantages, such as fast growth, easy genetic manipulation, low-cost culture media, and well-understood genetics. Therefore, it could be engineered rapidly, providing a fast system for initial laboratory-scale validations by our team.
2. The Saccharomyces cerevisiae modification strategy is valued for its fast growth, robustness, large scale capacity, and biosafety.
3. Aspergillus oryzae is an exceptional and central organism in this project due to its robust capacity for protein synthesis, and secretion making it an attractive host for expressing proteins. A standout feature of our approach is Aspergillus oryzae, a powerhouse microbe that secretes high levels of protein in both liquid and solid-state fermentation, unlocking scalable peptide production. By exploring its potential to adpated to lignocellulosic feedstocks, we created a sustainable and circular production model using orange peel residues from juice industry.

b. TRY (Titer, Rate and Yield)

Success hinges on titer, rate, and yield (TRY), which are essential metrics to decide whether a biotechnological process can scale from the lab to industry. Guided by this framework, we analyzed different strategies for producing the antimicrobial peptide CTX in bacteria, yeast, and filamentous fungi. By comparing not only technical performance but also cost-effectiveness, we showed how CTX can move beyond proof-of-concept and become a viable, sustainable solution to meet market demand.

The E.coli cultivated in liquid fermentation shows the lowest titer (2 mg/L), indicating limited capacity to produce the CTX. The production rate is also slow (0.6 mg/L/day), meaning the process takes a long time to reach significant concentrations. In addition, the yield relative to glucose consumption is low (0.2 mg/g), reflecting poor metabolic efficiency.

In another way, also cultivated in the liquid fermentation model, Saccharomyces cerevisiae titer increases to 12 mg/L and the production rate also improves, reaching 4 mg/L/day. The yield, expressed relative to biomass (15 mg/g), indicates that this microorganism converts a significant portion of cell growth into product.

Unexpectedly, the filamentous fungus Aspergillus oryzae cultivated in liquid fermentation showed slightly lower performance compared to Saccharomyces cerevisiae. In this system, A. oryzae reached a reasonable titer of 10 mg/L with a production rate of 1.6 mg/L/day, indicating a slower process than the yeast. Its glucose yield (1 mg/g) outperformed E. coli but remained inferior to the biomass conversion efficiency observed in S. cerevisiae.

However, Aspergillus oryzae cultivated in a solid-state fermentation using orange peel residues demonstrated the strongest overall performance: the highest titer (40 mg/L) and the fastest production rate (6.6 mg/L/day), reflecting strong time efficiency. The yield, expressed relative to residue (830 mg/kg), highlights the use of low-value substrates, which represents an advantage in terms of sustainability and cost reduction.

In this context, the capacity for peptide production has been demonstrated across different microorganisms and production systems. Among the four models, the Aspergillus oryzae cultivated in a solid-state fermentation model stands out as the most promising for industrial applications, combining high titer, high production rate, and the ability to use residues as feedstock, reducing costs and increasing sustainability.

c. Circular economy potential

Every year, the Brazilian orange juice industry generates millions of tons of residues during juice extraction, with half of the citrus fruit is discarded during the manufacture of juice generating large amounts of residues. These by-products are traditionally destined for secondary uses (LDC), such as a raw material for pectin extraction or low value cattle feed. However, the applications are poorly profitable, remaining a largely underexplored potential. Our team saw in this challenge an opportunity: to turn citrus waste into a resource for sustainable peptide production. By harnessing the natural ability of Aspergillus oryzae to grow on complex substrates and secrete large amounts of amylase enzymes, we aimed to establish a low-cost and eco-friendly system for CTX production through our AmyABC–CTX coupling strategy. To achieve this, we explored the use of solid-state fermentation (SSF) with orange peel residues as the growth substrate.

Brazil dominates the global orange juice trade, accounting for nearly 70% of worldwide production, with output projected to reach 1.01 million metric tons in the 2024/25 season, primarily supplying international markets. However, approximately half of the citrus fruit is discarded during juice manufacture, generating vast amounts of residue. These by-products, derived mainly from juice and pectin extraction, are typically of low quality and limited commercial value, which underscores the need for innovative and sustainable valorization strategies.

We designed a strategy to produce the CTX in Aspergillus oryzae by coupling its secretion with native amylases, achieving an estimated titer of 830 mg of CTX per kilogram of orange peel residue. We further validated that filamentous fungi can grow efficiently and secrete high levels of amylases when cultivated in a simple nitrogen medium supplemented with orange peels. These results highlight the potential of using this readily available agro-industrial substrate for low-cost CTX production, with the prospect of returning the peptide to citrus farms, either to combat greening disease or to protect fruits during post-harvest storage.

According to the National Solid Waste Plan, orange cultivation generates 8.8 million tons of waste annually in Brazil, highlighting the vast availability of this residue for use in solid-state fermentation processes. Based on our estimation, the CTX demand to address citrus greening in the national market alone would exceed 3 tons per year. To cover the 40 million tons of fruits destined for fresh consumption we estimated a demand of approximately 20 tons of CTX per year, based on the maximum permitted dosage of 5 mg/kg of imazalil in Brazil. Considering our yield of 830 mg of CTX per kilogram of orange peel residue, meeting this demand would require the processing of approximately 3.6 million kilograms (3,614 tons) of residues, a small fraction of residues generated annually in Brazil.

The integrated approach, which couples cutting-edge synthetic biology with circular economy principles, not only ensures cost-effectiveness but also reduces environmental impact, positioning the solution at the intersection of technological innovation and entrepreneurial viability. We also developed a guie to circularity which you can access below:

d. Competitive Advantage Matrix

Direct competitors are those actively engaged in the development of AMPs for citrus farming. These include companies that already operate with AMP technologies or biotechnological strategies focused on controlling plant pathogens.

• Invaio Sciences, based in Cambridge (USA), acquired in 2023 the startup Peptyde Bio, specialized in the discovery and development of broad-spectrum antimicrobial peptides for phytopathogen control. Their two main research areas are: (1) the use of artificial intelligence and machine learning for the identification and design of biological assets (Discovery Engine), and (2) the programming and implementation of Biological Delivery Systems aimed at improving delivery and field performance of active compounds, which biodegrade within 14 days of application. In citrus farming, particularly in the fight against Greening, the company launched the Trecise™ technology in Florida (USA). This system injects directly into the tree’s vascular system, reducing chemical use and increasing yields by an average of 30% in treated plants. In 2024, Invaio also established partnerships in Brazil, including a collaboration with Louis Dreyfus Company, aiming to bring this technology into Brazilian orchards and enable large-scale commercial adoption.

Despite the progress made by this competitor, there are still technical barriers that represent opportunities for our project. For example, the stability and half-life of AMPs in field conditions and the efficiency of their delivery into plant tissue remain critical challenges. Our system proposes a synthetic biology strategy for the large-scale production of AMP, specifically CTX, aimed at controlling the major pests and pathogens that threaten citrus chain, including Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum).

Indirect competitors are not directly focused on AMP development but offer alternative biocontrol or plant protection strategies that compete for the same target market. They appeal to growers seeking to reduce synthetic chemical use while boosting productivity and crop preservation.

• Koppert Biological Systems, a dutch company with more than 50 years of global operations, is a leader in biological solutions for pest and disease control. In the biofungicide segment, products like Trianum-P and Trianum-G, based on Trichoderma harzianum, are effective against soil-borne diseases such as Pythium, Rhizoctonia, and Fusarium, both in open fields and protected environments. In Brazil, Koppert has consolidated its portfolio around large crops like soybean, corn, and sugarcane, integrating solutions from seed treatment to drone-based applications, fully compatible with other agricultural practices.

Another competitor, Certis Biologicals, a subsidiary of Mitsui & Co., develops microbial-based biocontrols, botanical extracts, and biochemicals. With its own production centers and registrations in over 50 countries, the company offers biofungicides such as Double Nickel, based on Bacillus amyloliquefaciens. This product targets diseases like rust and leaf spots in crops including corn, soybean, and peanut, applicable both foliarly and in soil across all growth stages.

Although not AMP-based, these competitors benefit from strong market acceptance and established reputations. However, they also face limitations inherent to their technologies, such as variable efficacy under different environmental conditions, the need for frequent reapplications, and reduced stability during storage and transport. These gaps create opportunities for AMP-based solutions, such as our project, to emerge as more targeted, stable, and scalable alternatives.

Future competitors include companies not yet developing AMPs for agriculture but with strong technological capabilities and R&D pipelines that could quickly pivot into this segment. Their expertise spans areas such as AI-driven bioactive discovery, genome editing, recombinant protein synthesis, and biocontrol formulation, making them well-positioned to seize emerging market opportunities around AMP technologies.

• Micropep Technologies, founded in France in 2016 by CNRS and the University of Toulouse, has developed Krisalix, a platform for designing micropeptides (10–30 amino acids) capable of modulating gene expression in plants without altering DNA permanently, thereby enhancing plant immunity. Their applications currently target biofungicides and bioherbicides. Notably, their MPD-01 peptide, derived from tomato, demonstrates effective disease control in soybean (Soybean rust), potato (Phytophthora), and grapevine (Plasmopara viticola), with up to 75% efficacy.
• AgroSustain, founded in 2018 in Switzerland by the University of Lausanne, created Afondo, a coating platform composed of edible emulsifiers and vegetable oils. Applied by dipping or spraying, Afondo slows water loss by up to 35%, reduces postharvest fungicide use by about 50%, and extends refrigerated storage life of produce by 2 to 4 weeks.

These companies already benefit from robust technologies, strategic partnerships, and access to funding. If they decide to explore AMP-based solutions for agriculture, they could adapt existing platforms and shorten the timeline from development to commercialization. For our project, this represents a monitoring priority, requiring continuous tracking of industry trends and competitor movements.

Beyond the related-peptide market, it is also essential to consider management practices that do not involve the direct treatment of Greening, Sour Rot, and Green Mold.

• Greening (Huanglongbing, HLB):

Current management relies on integrated practices. In low-incidence areas, growers remove symptomatic trees, monitor orchard edges intensively, and apply insecticides to control Diaphorina citri, the main vector. Biological control agents are also used to sustainably reduce psyllid populations. For infected trees, foliar sprays with nutrients and phytohormones, along with soil pH correction, help mitigate symptoms and prolong tree lifespan. In the U.S., antibiotics such as streptomycin and oxytetracycline have been applied since 2019, although long-term health and environmental risks remain unresolved.

• Green Mold (Penicillium digitatum):

Management typically involves fungicide application, mainly imazalil and thiabendazole, but resistance and safety concerns limit their effectiveness. Alternative methods under study include carbonates, chlorides, natural extracts, hydrothermal treatments with spore removal systems, and ultraviolet radiation. These approaches highlight the urgent need for new, more efficient solutions.

• Sour Rot (Geotrichum candidum):

Control is particularly challenging during storage and transport. Fungicides effective against other molds (imazalil, thiabendazole, pyrimethanil, fludioxonil) do not work against G. candidum. Melanite has been approved in the EU and U.S. but not tested under Brazilian conditions. Sodium ortho-phenylphenate (SOPP) reduces symptoms but damages fruit peel and poses carcinogenic risks. Guazatine is one of the few effective fungicides, but it is banned in several countries, including Brazil, due to safety concerns. This lack of registered and reliable treatments makes the development of safe alternatives an urgent priority.

e. Risk Matrix

The Risk Matrix is a management tool that allows the identification, classification, and prioritization of potential risks to which a company is exposed, based on two fundamental axes: the probability of the event occurring and the impact if that event materializes. In its typical structure, risks are mapped in a table or chart in which each cell represents a combination between a probability level (low, medium, and high) and an impact level (also in classes such as insignificant, moderate, and catastrophic). This representation makes it possible to visualize which risks demand immediate attention, which should be monitored, and which may be acceptable, given their criticality.

By mapping possible events, our project becomes proactive, not only reacting to crises but also anticipating problematic scenarios. This allows us to develop mitigation and contingency plans or even avoid certain ventures if the perceived risk is catastrophic.

1. Team Description

Our team is a multidisciplinary group of 22 undergraduate and graduate students with backgrounds in biotechnology, biology, mathematics and physics.

Our scientific front is responsible for validating our solution and developing the process to produce it. Using a design, build and test method, we created and evaluated different production strategies to overcome challenges, such as the peptide's toxicity to the producing microorganism. As a result, we proved that the molecule works against the pathogens and that its biological production is viable, making the project technically safer for the next stages.

Our Modeling team is responsible for translating complex biological systems into decision-support computational tools that function as a virtual laboratory. Applying principles from computational and statistical physics, we developed a model that simulates disease dynamics and the effect of therapeutic interventions. All of our work is delivered with open-source code and detailed documentation, accelerating the development timeline and optimizing the allocation of research resources.

Our Hardware team operates across the full product development cycle, transforming a complex problem into a physical prototype. The process involves integrating advanced hardware, including nanotech sensors and custom circuits, with dedicated firmware and a machine learning pipeline to turn raw signals into diagnostics. As a result, we delivered a functional, data-validated prototype, establishing a modular and adaptable detection platform for new challenges.

Our Human Practices team is responsible for ensuring our technology aligns with real-world market needs, regulatory landscapes and stakeholder expectations. Using strategic tools like stakeholder mapping and the Power vs. Interest Matrix, we conducted engagement with players across the citrus value chain, including producers, industry and government bodies. This dialogue was a part of our iterative design process, ensuring the project's alignment with market demands and industry expectations.

Finally, our Entrepreneurship team brings together a unique blend of scientific insight, business acumen, and strategic vision. With members experienced in biotechnology companies, innovation programs, and project management, the group combined technical expertise with a strong understanding of market dynamics. This diversity allowed us to identify the technological and commercial potential of our solution, build a solid business model, and conduct a detailed market analysis that revealed key opportunities across the citrus value chain.

Also is valid to consider our strategic localization not only in citrus belt region in Brazil, but also at Unicamp. Here, we established key collaborations, such as with LaBioQui, led by Professor Taícia Fill, an expert in post-harvest citrus pathogens. Through this partnership, we were able to test CTX against Geotrichum candidum and Penicillium digitatum. As a result, we demonstrated that the molecule is effective against these pathogens and that its biological production is viable, making our project technically safer for the next stages. In addition, we have the support of LEBIMO, coordinated by Professor Dr. André Damasio, a laboratory specialized in protein production using the filamentous fungus Aspergillus oryzae. With access to both the laboratory facilities and their technical expertise, we can advance the development of the liquid-state fermentation process for CTX production using A. oryzae. Building upon these developments, our location at Unicamp also offers access to specialized facilities for solid-state fermentation using orange peel residues, such as the laboratory coordinated by Professor Gabriela Alves Macedo (Head of Bioprocess and Biochemistry of Bioactive Compounds). This strategic advantage allows us to expand our approach toward a circular economy model, cultivating A. oryzae in solid-state fermentation with citrus residues. Finally, we have also established partnerships with research institutions beyond our university, such as the Centro de Citricultura Sylvio Moreira, coordinated by Professor Alessandra Alves de Souza, a citrus-specialized research center that enables us to carry out CTX testing against Candidatus Liberibacter asiaticus, the bacterium responsible for citrus greening disease.

Overall, our team stands out not only for its academic excellence but also for its leadership, creativity, and commitment to generating real-world impact. In just a few months, these combined qualities have translated into concrete achievements, showcasing the group’s ability to transform ideas into sustainable, high-impact innovations.

2. Interested parts and Stakeholders

Effectively managing all key parties by a venture is essential for the success of a business. These groups are collectively known as stakeholders, encompassing everyone from directors, investors, and employees to customers, suppliers, regulatory agencies, and the general public. Understanding these different stakeholders and their interconnections allows ventures to build trust, strengthen collaboration, and ensure that decision-making aligns with both business goals and social responsibility.

To guide this process, we used the Power vs. Interest Matrix, a tool that helps visualize how different actors relate to a project. In this matrix, “power” represents the ability of a stakeholder to influence outcomes, while “interest” reflects how much they are affected by or invested in the solution. By positioning our stakeholders in this framework, we could plan tailored strategies for each group, ensuring that our time and resources were invested where they could generate the greatest impact. To achieve this, the Stakeholder Matrix serves as a supporting tool to identify and classify the different stakeholders involved based on the influence, power, interest, impact, support, and importance each represents within the project's scope.

It is possible to interact with the chart to better understand how we plan approaches with each of the mapped stakeholders.

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Typically, these engagement levels are divided into four categories, which represent the quadrants of the matrix:

High Power, High Interest (Actively Engaged)

These are the essential actors without whom the project cannot advance. In our case, rural producers, sponsors, and partner companies fall into this category, since their insights and needs directly shape the scientific and practical direction of Pepcitrus. Their feedback is not just useful; it is critical.

High Power, Low Interest (Keep Satisfied)

This group includes regulators, legal experts, and institutions whose approval is necessary for implementation but who are not involved in the daily progress of the project. Keeping them satisfied involves providing reliable updates and ensuring compliance, so they remain supportive without feeling overwhelmed.

Low Power, High Interest (Keep Informed)

This group includes the general public, consumers, and student communities who, although they cannot directly change the project’s course, can be affected by its outcomes. Ensuring they are informed builds trust, fosters social legitimacy, and encourages acceptance of biotechnological innovations.

Low Power, Low Interest (Monitor)

These are peripheral actors with limited influence and stake in the project. They only require occasional monitoring to ensure no overlooked factor becomes a future risk.

During the project, we had the opportunity to connect with and learn from entrepreneurship specialists, presented below. These meetings were fundamental in building our brainstorming process and developing the business plan. The support of experienced professionals was essential to broaden our strategic vision, guide critical decisions, and identify new growth opportunities that might not have been perceived without this exchange. A more detailed description of the interviews conducted with these specialists can be found on the integrated human practices page.

a. INOVA UNICAMP

INOVA's mission is to connect the university with society, fostering research, teaching, and the advancement of knowledge through collaborative networks. During our conversation, INOVA emphasized the importance of conducting economic analyses to assess tangible impact, and with this perspective, we were able to look beyond the scientific scope and incorporate tools that demonstrate the real value generated for both the environment and society. By grounding our initiatives in robust evidence and reliable data, we learned how to communicate the project's benefits in a way that resonates not only with the academic community but also with potential investors, partners, and end users.

b. PEPTIDUS BIOTECH

Peptidus Biotech is a company focused on developing solutions using generative artificial intelligence and structural biology for the optimization of bioactive peptides. These solutions stand out for being sustainable, efficient, and free of residues harmful to the environment or human health. Our contact with them allowed us to explore AMP production while optimizing costs and aligning scientific advances with market needs. For example, discussions on scalability and production accessibility reinforced the importance of designing solutions that can truly reach the agricultural sector, aiming for a cost of around 50 cents per dose, being non-toxic, and ensuring ease of application.

c. CAFÉ COM BIOECONOMIA

The “Café com Bioeconomia” podcast by SENAI RIO Brazil aims to boost innovation in bioeconomy through discussions on topics of broad national interest, fostering connection, dialogue, and highlighting opportunities and challenges for businesses. Our interaction provided a strategic opportunity to promote our project and connect with stakeholders in the business sector.

The podcast introduced us to the Technical and Economic Feasibility Study (EVTE) as an essential tool to present the market potential of our project to companies and sponsors. This highlighted the need to design our project not only as a scientific solution but also as a business opportunity supported by economic data and strategic alignment. In addition, we focused on understanding Brazil's Biodiversity Law, which must be considered in the use of natural peptides. This perspective reinforced the importance of integrating legal and market analyses into our entrepreneurial journey.

d. DOUGLAS MARTINS VERONEZ (EMERGE)

Emerge Brasil is a consulting firm specialized in science-based innovation, known as deep tech, that has been operating since 2017 with the mission of transforming scientific research into market-ready solutions in Brazil. During our discussion, we reflected on the main challenges faced by companies adopting deep techs in the country, including regulatory barriers and scalability issues, and explored how to evaluate the feasibility and long-term impact of biotechnological solutions like ours. We also learned about the importance of aligning innovation with sustainability goals and the need to anticipate strategies to mitigate risks during the implementation phase.

An important highlight was the emphasis that “having a good technology is not enough; it is necessary to know how to communicate its value to the market,” reinforcing the importance of considering business models, technical viability, and regulatory impact from the very beginning of project development. As a practical outcome, we enrolled in the course offered by EMERGE to acquire essential knowledge in scientific innovation and entrepreneurship and later requested mentorship to help us build our business case.

e. CITRUS FARMERS

From smallholders to large-scale suppliers, citrus producers struggle with high economic losses caused by persistent diseases. Current chemical solutions are ineffective, unavailable for certain pathogens, or increasingly restricted by regulations. We observed this scenario during our conversations with large producers such as AlfaCitrus and small farmers we met throughout our fieldwork, including at the largest citrus fair in Latin America (Expocitros).

Beyond Greening, the main challenge identified for Alfacitrus was the high incidence of Geotrichum candidum, which causes Sour Rot. This post-harvest disease affects fruits during storage and transport, and current fungicides do not control it. As a major supplier to the Brazilian domestic market, Alfacitrus incurs significant losses at this stage of production when each fruit carries a high added value due to harvesting, sorting, and logistics costs. Infections at this stage can compromise entire batches, forcing the disposal of tons of fruit destined for national distribution. The lack of effective, environmentally safe fungicides increases operational risks and dependency on ineffective, outdated treatments.

For small farmers, Greening, which is transmitted by the psyllid Diaphorina citri, remains the primary concern. Since they have limited land and fewer plants, eradicating infected trees results in greater production losses for them than for large-scale producers. A single infected tree can represent years of lost investment and serve as a reservoir for the greening bacteria to spread. Furthermore, the absence of preventive solutions forces many to rely on expensive chemical products that only temporarily halt the inevitable spread. This scenario creates a cycle of economic fragility. Families depend on each harvest to sustain their livelihoods yet they face escalating input costs and declining productivity. Tiago and his father are an example of this, they are one of the last families to maintain the tradition of citrus farming in their region, despite all the threats.

The burden extends beyond the farm gate for both groups. The constant need for pesticide application raises operational costs, degrades soil health, and contributes to environmental contamination. This scenario reveals the dual reality of the citrus sector: small farmers struggle to survive, while large producers strive to remain competitive in the national market. Beyond farm-level economics, these losses ripple through the food chain, reducing the supply of fresh fruit and juice for consumers and raising broader concerns about food security.

3. Pitch Deck – Pepcitrus

To communicate our vision effectively, we developed a Pitch Deck that summarizes the problem, our innovative solution, the market potential, and the societal impact of Pepcitrus. A Pitch Deck is an essential tool because it translates complex scientific and technical work into a clear, compelling, and accessible narrative, allowing us to engage with a broad range of stakeholders.

To ensure the technical feasibility and market success of the Pepcitrus project, we are implementing a comprehensive development strategy that integrates Technology Readiness Level (TRL) analysis, techno-economic assessment (TEA), life cycle assessment (LCA), and roadmap planning. This combined approach enables a systematic evaluation of our solutions across technological, economic, environmental, and strategic dimensions. Specifically:

1. Roadmap planning consolidates these insights into a strategic timeline, aligning R&D milestones with market entry opportunities.
2. Technology Readiness Level (TRL) provides a structured framework to track and advance technological maturity, minimizing risks during scale-up.
3. Life cycle assessment (LCA) maps environmental impacts across the value chain, reinforcing sustainability and regulatory alignment.
4. Techno-Economic Assessment (TEA) evaluates production costs, scalability, and market competitiveness, ensuring financial viability.

By integrating these analysis, we aim not only to optimize implementation and scalability but also to anticipate potential impacts, reduce risks, and ensure evidence-based decision-making, thereby strengthening the strategic relevance and value proposition of Pepcitrus within the citrus industry.

1. Roadmap planning and Technology Readiness Level

Technology Readiness Level (TRL) is a methodology for assessing the maturity of a technology by classifying its progress from basic research to full operational deployment in the market. Different sectors, such as the pharmaceutical or information technology industries, often adapt the TRL scale to address the particularities of their respective innovation processes. Despite these variations, the scale is universally composed of nine levels, generally grouped as follows:

1. TRL 1-3: Basic Research and Development. This phase corresponds to basic research, the formulation of technological concepts, and experimental proof-of-concept.
2. TRL 4-6: Technology Development and Validation. This involves the validation of components in a laboratory environment and subsequently in a relevant environment (simulated or real) to demonstrate the technology's viability.
3. TRL 7-9: Demonstration and Deployment. This refers to the demonstration of the system prototype in an operational environment, qualification of the final system, and ultimately, successful commercial production and operation.

In Pepcitrus, our roadmap transforms scientific insights into a clear strategic timeline, guiding the project from laboratory discoveries to field applications. By aligning R&D milestones with regulatory pathways and market opportunities, we ensure that peptide-based solutions like CTX move seamlessly from proof of concept to scalable deployment in citrus production. This roadmap not only directs our innovation efforts but also secures a pathway for sustainable impact in the bioinputs market.

Roadmap Planing:

2. Life Cycle Assessment (LCA)

Integrating Life Cycle Assessment (LCA) into the early stages of process development allows teams to anticipate a technology’s environmental footprint long before it reaches commercial scale. LCA helps identify critical “hotspots,” such as energy-intensive sterilization or high enzyme consumption, guiding R&D toward process optimizations that deliver both economic and ecological benefits. By modeling emissions, energy use, and water demand per gram of CTX, we can set measurable sustainability targets and prioritize innovations—such as renewable energy sourcing or enzyme recycling—that reduce impacts over time. In the long term, a cradle-to-gate LCA provides valuable insights for regulatory compliance and market positioning. In this sense, our study performed a cradle-to-gate Life Cycle Assessment covering the main steps involved in CTX production across three expression platforms: E. coli, S. cerevisiae, and A. oryzae. To estimate factors such as raw material use, energy demand, equipment needs, and depreciation, we used data from an EMBRAPA Technical Circular and technical assumptions guidelined by ISO 14044.

Integrating LCA into development plans helps anticipate the environmental footprint of a process long before commercial scale is reached. It highlights “hotspots” such as high-energy sterilization or enzyme usage, guiding R&D efforts toward process improvements that deliver both economic and ecological benefits. By modeling emissions, energy demand, and water use per gram of CTX, we can set clear sustainability targets and prioritize innovations, such as renewable energy sourcing or enzyme recycling, that will reduce impacts over time.

As illustrated in the system diagram, the LCA boundaries span from raw material extraction (“cradle”) to the point where the purified CTX product leaves the production facility (“gate”). This scope quantifies key environmental indicators—greenhouse gas emissions, energy consumption, and water use—while excluding downstream stages such as product distribution, use, and end-of-life. Such an approach is particularly suited to early-stage biotechnological processes, where the focus is on optimizing production efficiency and sustainability before commercialization. More detailed definitions of the objective, scope, limitations, and assumptions are provided in the following spreadsheet.

Thus, the LCA revealed that E. coli has the highest environmental burden, with approximately 135 kg CO₂e per gram of CTX and energy consumption of 1,800 MJ/g, driven by the need for 50 production batches per year. In contrast, A. oryzae in solid-state fermentation achieves the lowest footprint, with roughly 30 kg CO₂e per gram and only 90 MJ/g of energy demand. S. cerevisiae and liquid fermentation of A. oryzae fall in between, reflecting intermediate titers and process efficiencies.

3. Techno-Economic Assessment (TEA)

A Techno-Economic Assessment (TEA) is a structured method used to evaluate whether a technology can be produced at scale with economic viability.It integrates technical parameters such as yield, production time, energy demand, and process complexity with financial indicators including capital expenditures, operating costs, and unit production cost. By merging these perspectives, TEA helps determine not only whether a process is technically possible, but also whether it can compete in a real market and generate sustainable profits. In the entrepreneurial context, an TEA provides the bridge between scientific discovery and market reality. It enables teams to identify cost drivers, estimate future selling prices, and prioritize research directions that offer the highest potential for cost reduction.

Comparative analysis of CTX production performance across different microbial hosts and cultivation strategies. Parameters include titer, production rate, yield, and scalability metrics expressed as batches per year and final annual output, considering a 10,000 L fermentation volume per batch.

The economic analysis revealed striking differences between hosts and cultivation strategies. While E. coli and S. cerevisiae resulted in prohibitively high production costs, over 1.5 million dollars and 650,000 dollars per kilogram of CTX, respectively, A. oryzae offered a clear advantage. In submerged fermentation the cost decreased to around 236,000 dollars per kg, but the most promising strategy was solid-state fermentation using citrus residues, which reduced the cost to approximately 57,000 dollars per kg. This drastic reduction is mainly attributed to the low substrate and energy requirements of solid fermentation, highlighting the relevance of circular economy approaches. Importantly, these calculations were based on a reference scale of a 10 m³ reactor. By increasing the work volume and number of reactors, combined with parallel batch operations, the process could be further optimized, reducing fixed costs per unit of product and improving overall economic feasibility. Such modular scalability makes the transition from lab to industry more realistic, since multiple mid-scale units can collectively reach production levels comparable to large facilities, but with greater flexibility and lower capital investment.

It is important to note that the current estimates do not account for the additional costs associated with purification resins and TEV protease, both of which are significant contributors in downstream processing. These factors would considerably increase the overall cost of production, reinforcing that the reported values represent optimistic lower bounds rather than final industrial estimates. More rigorous analyses will be required to capture the full economic landscape, but the values presented here serve as indicative estimates to highlight the order-of-magnitude differences between strategies and to demonstrate that CTX production can indeed become economically feasible under optimized conditions

Taken together, the LCA and TEA analyses show that economic and environmental performance are closely aligned: the platform with the best cost profile (A. oryzae solid-state fermentation) is also the most sustainable. This convergence provides a powerful foundation for long-term development, where increasing titers, adopting renewable energy, and implementing circular feedstocks (such as citrus by-products) can further lower costs and carbon intensity. By embedding LCA and TEA into the early stages of planning, our team ensures that CTX production will not only reach commercial feasibility but also meet future demands for low-impact environment.

3. Assessing Direct and Indirect Impacts

When developing a product, it is essential to evaluate both its positive and negative long-term impacts as part of a fully integrated solution. In the case of our large-scale antimicrobial peptide (AMP) production platform our goal is to control the main pests and pathogens threatening the citrus chain, including Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum).

Unlike conventional chemical pesticides, our biotechnology-based solution employs sustainable and safe mechanisms to effectively suppress these diseases. It significantly reduces the need for antibiotics such as oxytetracycline, widely used in citrus orchards to control Greening but associated with environmental and public health concerns.

The fermentation-based production of CTX using genetically modified microorganisms provides a more sustainable and efficient alternative to traditional chemical synthesis, enabling large-scale application and manufacturing. Moreover, by overcoming the complexity of microbial AMP production through a synthetic biology strategy that couples CTX to highly expressed host proteins, we open new avenues for biotechnological manufacturing. This approach reduces dependence on toxic chemicals, lowers energy consumption, minimizes raw material use, and allows scalable production.

Together, these advances position our technology as a high-impact alternative that bridges cutting-edge science with tangible environmental, economic, and social benefits—addressing the urgent need for innovation in citrus protection.

Furthermore, the Pepcitrus project is directly aligned with six of the United Nations Sustainable Development Goals (SDGs), organized into four key dimensions

1. Food:

a. SDG 2 - Zero Hunger and Sustainable Agriculture: In a country that produces abundantly yet faces food insecurity, innovation becomes essential to bridge the gap between production and access. Our project introduces a novel strategy for CTX production in multiple microbial hosts and its application against pre- and post-harvest citrus diseases. By reducing post-harvest losses and improving field productivity, Pepcitrus contributes to greater food availability and stability.

b. SDG 12 - Responsible Consumption and Production: Addressing food waste is fundamental to achieving balance in the global food system. By rethinking value chains and reducing inefficiencies, our approach promotes a more conscious and circular bioeconomy.

2. Industry:

a. SDG 8 - Decent Work and Economic Growth: With millions trapped in informality, the challenge is not just to create income, but to ensure dignified and sustainable opportunities. This scenario demands new ways of building value, capable of transforming vulnerability into collective strength.

b. SDG 9 - Industry, Innovation and Infrastructure: The future of production cannot rest solely on machines, but on knowledge and collaborative networks. To modernize is also to reinvent fragile processes, preparing them to withstand crises and create long-lasting impact.

3. Partnerships:

a. SDG 17 - Partnerships for the Goals: None of these challenges can be solved in isolation. Strength lies in cooperation, in the exchange of diverse realities and knowledge, where local and global alliances become the very foundations of structural change.

Further details on these contributions can be found on our Sustainable Development page.

Equally important is the consideration of potential negative impacts. The high cost and import dependence of certain feedstocks may limit market competitiveness when compared to faster and cheaper chemical or biological alternatives. These challenges highlight the need for strategic planning in regulatory navigation, process optimization, and cost reduction to ensure large-scale adoption without compromising sustainability and efficacy.

In conclusion, the Pepcitrus project embodies a balance between sustainability, technological innovation, and market viability. By reducing reliance on conventional antibiotics and implementing environmentally responsible CTX production, it addresses critical challenges in citrus agriculture while fostering cleaner and more efficient practices. At the same time, regulatory and economic barriers call for continued strategic development to ensure global scalability. Overall, Pepcitrus aligns innovation with social and environmental responsibility, positioning itself as a transformative biotechnology capable of delivering long-term impact across the entire citrus value chain.