The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

ABSTRACTArthrospira platensis(commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina’s genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

ABSTRACTBackgroundArthrospira platensis (commonly known as spirulina) is a promising new platform for low-cost manufacturing of biopharmaceuticals. However, full realization of the platform’s potential will depend on achieving both high growth rates of spirulina and high expression of therapeutic proteins.ObjectiveWe aimed to optimize culture conditions for the spirulina-based production of therapeutic proteins.MethodsWe used a machine learning approach called Bayesian black-box optimization to iteratively guide experiments in 96 photobioreactors that explored the relationship between production outcomes and 17 environmental variables such as pH, temperature, and light intensity.ResultsOver 16 rounds of experiments, we identified key variable adjustments that approximately doubled spirulina-based production of heterologous proteins, improving volumetric productivity between 70% to 100% in multiple bioreactor setting configurations.ConclusionAn adaptive, machine learning-based approach to optimize heterologous protein production can improve outcomes based on complex, multivariate experiments, identifying beneficial variable combinations and adjustments that might not otherwise be discoverable within high-dimensional data.