Synthetic biology platform for engineering heat- and acid-stable natural sweet proteins

Overview

This solution offers a synthetic biology-driven approach to stabilising natural sweet proteins for use in food and beverage applications. Natural sweet proteins such as thaumatin and brazzein are thousands of times sweeter than sugar but typically degrade under industrial processing conditions involving high heat and low pH. By combining ancestral sequence reconstruction (reverse evolution) with directed evolution (forward evolution), the platform systematically explores vast regions of protein sequence space to identify variants that retain sweetness and structural integrity under harsh conditions. The result is a pipeline for producing next-generation sweet proteins suitable for formulation in acidic beverages, baked goods, and other processed food products.

Technical specifications

Core methodology:

• Ancestral sequence reconstruction is used to resurrect ancient thaumatin-like proteins from present-day sequence data; ancestral proteins are intrinsically tolerant of high temperatures and low pH
• Directed evolution applies iterative rounds of genetic diversification and selective pressure to optimise both thaumatin ancestors and brazzein variants (for which ancestral reconstruction is not feasible due to limited sequence relatives)
• High-throughput screening evaluates tens of thousands of protein variants for heat and acid stability, where unstable proteins unfold and precipitate while stable proteins remain soluble
• A yeast-based synthetic biology reporter assay tests whether stabilised variants successfully activate the T1R2/T1R3 human sweet taste receptor
• mRNA display is used to identify sequences of surviving soluble proteins after each round of heat treatment
• Further targeted modifications of charged, solvent-exposed residues enhance acid stability in lead candidates

Key advantages:

• Leverages two complementary protein engineering strategies to address both large (~200 amino acid) and small (~50 amino acid) sweet protein classes
• Validated precedent: ancestral proteins have been shown to tolerate temperatures 30–50 °C higher than present-day counterparts
• Heat-stable scaffolds can be further engineered for additional properties without loss of function

Technology readiness level

The platform is at an early-to-mid stage of development. Ancestral reconstruction methodology has been validated, with dozens of ancient proteins already resurrected and confirmed to exhibit superior thermal stability compared to modern variants. The yeast-based T1R2/T1R3 sweet taste receptor reporter assay is established and operational. Future validation work will focus on synthesising and testing ancestral thaumatin variants for receptor activation before and after heat treatment, evolving brazzein variants through successive heat-selection rounds, and applying further directed evolution to enhance acid stability in confirmed sweet, heat-stable lead candidates.