Strain engineering and bioprocess development for bio-based production of porphyrins

Abstract

Bio-based production using microbial cell factories has emerged as a transformative approach to addressing the limitations of petrochemical processes, offering renewable, sustainable, and environmentally friendly alternatives for manufacturing valuable chemical compounds. Among various microbial systems, Escherichia coli (E. coli) has become a popular and versatile host for biomanufacturing due to its rapid growth, genetic tractability, and extensive history of industrial use. Through advances in synthetic biology, genome engineering, and metabolic engineering, E. coli can be tailored to produce a wide array of chemicals, including structurally complex compounds like porphyrins. Porphyrins and their derivatives, such as heme and chlorophyll, are critical for various biological and industrial applications, ranging from pharmaceuticals and diagnostics to renewable energy solutions. Despite their significance, challenges such as pathway bottlenecks, feedback inhibition, and intracellular toxicity of intermediates hinder microbial production of porphyrins. This thesis addresses these challenges by implementing integrated engineering strategies to enhance porphyrin biosynthesis in E. coli and establish a robust framework for scalable production. A foundation of this work was the development of a genome engineering toolkit that integrates CRISPR-Cas9 and transposon-based methods. This system allowed for site-specific insertion of heterologous genes and precise inactivation of endogenous genes, achieving editing efficiencies exceeding 90%. Such flexible manipulation of the E. coli genome facilitated the construction of optimized strains for biomanufacturing. For example, the toolkit enabled the creation of a plasmid-free strain capable of producing polyhydroxyalkanoates, demonstrating its potential for industrial applications and providing the foundation for metabolic engineering efforts targeting porphyrin biosynthesis. For the subsequent part of our thesis, the biosynthesis of uroporphyrin (UP), a key precursor for heme, was enhanced by implementing the Shemin/C4 pathway in E. coli. Strategies to increase intracellular succinyl-CoA availability and express a synthetic operon containing genes such as hemA, hemB, hemC, and hemD led to UP titers of 901.9 mg/L under batch bioreactor conditions. Furthermore, most of the UP produced was secreted extracellularly, simplifying downstream purification and demonstrating the feasibility of large-scale production. These advancements highlight the effectiveness of pathway optimization in overcoming metabolic bottlenecks. We used the information obtained from previous chapter to enhance coproporphyrin (CP) biosynthesis. Dual synthetic operons controlled by strong promoters regulated key pathway genes, including hemA, hemB, hemD, hemE, and hemY. Bioreactor cultivation of the engineered strains using glycerol as the primary carbon source under aerobic conditions led to CP titers of up to 353 mg/L with minimal byproduct formation. To the best of our knowledge this study marked the first targeted bio-based production of CP in E. coli, laying the groundwork for its industrial-scale synthesis and emphasizing the importance of precise gene regulation in pathway optimization. Addressing the complexities of heme biosynthesis required a novel two-step strategy integrating in vivo and in vitro approaches. Engineered E. coli strains expressing the coproporphyrin-dependent (CPD) pathway produced ∼85 mg/L of coproheme and ∼18 mg/L of heme in vivo. However, intracellular heme accumulation posed significant toxicity challenges due to limited secretion into the extracellular medium. These challenges were mitigated by developing an optimized in vitro enzymatic conversion process, achieving a 77.2% reaction yield for the conversion of coproporphyrin III to coproheme and a 45.8% yield for the conversion of coproheme to heme. This integrated approach bypassed intracellular toxicity, enabling controlled and scalable production while addressing key bottlenecks in microbial production systems. In our final study, to further enhance porphyrin biosynthesis, strategies were developed to mitigate reactive oxygen species (ROS)-induced stress and redirect dissimilated carbon flux toward type-III porphyrin biosynthesis. Antioxidant supplementation with ascorbic acid (up to 1 g/L) improved the UP-III/UP-I ratio from 0.62 to 2.57, enhancing the production of type-III porphyrins. Additionally, overexpression of ROS-scavenging genes such as sodA and kat significantly increased porphyrin yields. Notably, overexpression of sodA alone resulted in a 72.9% increase in total porphyrin production, reaching titers of 1.56 g/L, while improving the UP-III/UP-I ratio to 1.94. These findings underscore the importance of addressing oxidative stress to optimize metabolic fluxes and enhance type-III porphyrin biosynthesis in E. coli. The study provides a practical platform for improving bio-based porphyrin production at industrial scales. Taken together, this thesis demonstrates the potential of integrating strain engineering, synthetic biology, and metabolic engineering to enhance porphyrin biosynthesis in E. coli. The innovative strategies developed provide scalable, sustainable, and economically viable solutions for producing porphyrins and their derivatives. These advancements open new avenues for industrial applications in pharmaceuticals, diagnostics, and renewable energy, establishing E. coli as a powerful platform for biomanufacturing complex biomolecules.