Genetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.
Berberine is an extensively used pharmaceutical benzylisoquinoline alkaloid (BIA) derived from plants. Microbial manufacturing has emerged as a promising approach to source valuable BIAs. Here, we demonstrated the complete biosynthesis of berberine in Saccharomyces cerevisiae by engineering 19 genes including 12 heterologous genes from plants and bacteria. Overexpressing bottleneck enzymes, fermentation scale-up, and heating treatment after fermentation increased berberine titer by 643-fold to 1.08 mg L-1. This pathway also showed high efficiency to incorporate halogenated tyrosine for the synthesis of unnatural BIA derivatives that have higher therapeutical potentials. We firstly demonstrate the in vivo biosynthesis of 11-fluoro-tetrahydrocolumbamine via nine enzymatic reactions. The efficiency and promiscuity of our pathway also allow for the simultaneous incorporation of two fluorine-substituted tyrosine derivatives to 8, 3’-di-fluoro-coclaurine. This work highlights the potential of yeast as a versatile microbial biosynthetic platform to strengthen current pharmaceutical supply chain and to advance drug development.
Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant natural products with extensive pharmacological properties, but the yield of BIAs from plant is limited. The understanding of BIA biosynthetic mechanism in plant and the development of synthetic biology enable the possibility to produce BIAs through microbial fermentation, as an alternative to agriculture-based supply chains. In this review, we discussed the engineering strategies to synthesize BIAs in Saccharomyces cerevisiae (yeast) and improve BIA production level, including heterologous pathway reconstruction, enzyme engineering, expression regulation, host engineering and fermentation engineering. We also highlight recent metabolic engineering advances in the production of BIAs in yeast.
Yeast has been a versatile model
host for complex and valuable
natural product biosynthesis via the reconstruction of heterologous
biosynthetic pathways. Recent advances in natural product pathway
elucidation have uncovered many large and complicated plant pathways
that contain 10–30 genes for the biosynthesis of structurally
complex, valuable natural products. However, the ability to reconstruct
ultralong pathways efficiently in yeast does not match the increasing
demand for valuable plant natural product biomanufacturing. Here,
we developed a one-pot, multigene pathway integration method in yeast,
named MULTI-SCULPT for multiplex integration via selective, CRISPR-mediated,
ultralong pathway transformation. Leveraging multilocus genomic disruption
via CRISPR/Cas9, newly developed native and synthetic genetic parts,
and fine-tuned gene integration and characterization methods, we managed
to integrate 21 DNA inserts that contain a 12-gene plant isoflavone
biosynthetic pathway into yeast with a 90–100% success rate
in 12 days. This method enables fast and efficient ultralong biosynthetic
pathway integration and can allow for the fast iterative integration
of even longer pathways in the future. Ultimately, this method will
accelerate combinatorial optimization of elucidated plant natural
product pathways and accelerate putative pathway characterization
heterologously.
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