Systematic development of a highly efficient cell factory for 5-aminolevulinic acid production

• Published in: Trends in biotechnology (Regular ed.) 2024-11, Vol.42 (11), p.1479-1502
• Main Authors: Zhou, Houming, Zhang, Chengyu, Li, Zilong, Xia, Menglei, Li, Zhenghong, Wang, Zhengduo, Tan, Gao-Yi, Luo, Ying, Zhang, Lixin, Wang, Weishan
• Format: Article
• Language: English
• Subjects: 5-Aminolevulinate Synthetase - genetics; 5-Aminolevulinate Synthetase - metabolism; 5-aminolevulinic acid; Acid production; Aminolevulinic acid; Aminolevulinic Acid - metabolism; artificial homeostasis; Bioreactors; Biosynthesis; Biotechnology; Carbon; Cell growth; collaborative optimization; Control systems; Coronaviruses; COVID-19; Dynamic control; dynamic regulation; E coli; Engineering; enzyme mining; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Homeostasis; Livestock; Metabolic Engineering - methods; Metabolism; Mining; Optimization; Oxidation-Reduction; Plant layout; Scale models; systems metabolic engineering; targets combination
• ISSN: 0167-7799; 1879-3096; 1879-3096
• DOI: 10.1016/j.tibtech.2024.06.004

Establishment of a highly efficient cell factory is imperative for 5-aminolevulinic acid (5-ALA) biomanufacturing.A streamlined workflow is described that enables highly efficient 5-ALA synthase mining.Genome-scale model-guided identification and combination of multiplex targets are reported.An artificial homeostasis was designed for dynamically responding to, and fine-tuning, redox status.Final collaborative optimization resulted in the highest 5-ALA biomanufacturing performance achieved to date. The versatile applications of 5-aminolevulinic acid (5-ALA) across the fields of agriculture, livestock, and medicine necessitate a cost-efficient biomanufacturing process. In this study, we achieved the economic viability of biomanufacturing this compound through a systematic engineering framework. First, we obtained a 5-ALA synthase (ALAS) with superior performance by exploring its natural diversity with divergent evolution. Subsequently, using a genome-scale model, we identified and modified four key targets from distinct pathways in Escherichia coli, resulting in a final enhancement of 5-ALA titers up to 21.82 g/l in a 5-l bioreactor. Furthermore, recognizing that an imbalance of redox equivalents hindered further titer improvement, we developed a dynamic control system that effectively balances redox status and carbon flux. Ultimately, we collaboratively optimized the artificial redox homeostasis system at the transcription level with other cofactors at the feeding level, demonstrating the highest recorded performance to date with a titer of 63.39 g/l for the biomanufacturing of 5-ALA. Graphical abstract [Display omitted] The economic viability of biomanufacturing 5-aminolevulinic acid (5-ALA) was successfully demonstrated in this study, showcasing excellent production performance. The titer reached a record-breaking 63.39 g/l at 44 h, representing the highest reported value to date, with the productivity of 1.44 g/l/h. Although the yield (0.384 mol/mol glucose) was lower than theoretically expected, the significant value of 5-ALA positions our developed cell factory competitively for efficient industrial-scale biomanufacturing. Therefore, no challenges unique to this compound can be identified for full-scale fermentation, particularly considering that our 5-ALA cell factory was derived from a widely utilized Escherichia coli strain. Currently, two primary approaches are used to produce 5-ALA: chemical synthesis and biomanufacturing. Microbial biosynthesis o