Artificial Biosynthetic Pathway for an Unnatural Terpenoid with an Iridiumcontaining P450

<div>Synthetic biology enables microbial hosts to produce complex molecules that are</div><div>otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these</div><div>molecules are limited to chemical reactions catalyzed by natural enzymes. The integration of</div><div>artificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks could</div><div>broaden the cache of molecules produced biosynthetically by microorgansms. We report the</div><div>assembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpene</div><div>producing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArM</div><div>into a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows that</div><div>synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce</div><div>molecules previously inaccessible to nature.</div>

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Unnatural Biosynthesis by an Engineered Microorganism with Heterologously Expressed Natural Enzymes and an Artificial Metalloenzyme

10.21203/rs.3.rs-130318/v1 ◽

2021 ◽

Author(s):

Jing Huang ◽

Zhennan Liu ◽

Brandon Bloomer ◽

Douglas Clark ◽

Aindrila Mukhopadhyay ◽

...

Keyword(s):

Synthetic Biology ◽

Biosynthetic Pathway ◽

Metabolic Networks ◽

Cultivation Conditions ◽

Complex Molecules ◽

Whole Cells ◽

E Coli ◽

Product Titer ◽

Artificial Metalloenzymes ◽

Artificial Metalloenzyme

Abstract Synthetic biology enables microbial hosts to produce complex molecules that are otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by chemical reactions catalyzed by natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically by microorganisms. We report an engineered microbial cell expressing a heterologous biosynthetic pathway, which contains both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. To create this hybrid biosynthetic organism, we engineered Escherichia coli (E. coli) with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, together with natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.

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Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515826113 ◽

2016 ◽

Vol 113 (12) ◽

pp. 3209-3214 ◽

Cited By ~ 97

Author(s):

Bradley Walters Biggs ◽

Chin Giaw Lim ◽

Kristen Sagliani ◽

Smriti Shankar ◽

Gregory Stephanopoulos ◽

...

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Metabolic Engineering ◽

Biosynthetic Pathway ◽

Complex Molecules ◽

E Coli ◽

Precursor Synthesis ◽

P450 Expression ◽

High Level

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

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Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

10.26434/chemrxiv.10333625.v1 ◽

2019 ◽

Author(s):

Thomas Siemon ◽

Zhangqian Wang ◽

Guangkai Bian ◽

Tobias Seitz ◽

Ziling Ye ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Synthetic Biology ◽

Chemical Synthesis ◽

Building Block ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Economical Method ◽

Englerin A ◽

Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

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Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

10.26434/chemrxiv.10333625 ◽

2019 ◽

Author(s):

Thomas Siemon ◽

Zhangqian Wang ◽

Guangkai Bian ◽

Tobias Seitz ◽

Ziling Ye ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Synthetic Biology ◽

Chemical Synthesis ◽

Building Block ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Economical Method ◽

Englerin A ◽

Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

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Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68123-2 ◽

1988 ◽

Vol 263 (29) ◽

pp. 14900-14905

Author(s):

J L Milner ◽

S Grothe ◽

J M Wood

Keyword(s):

Escherichia Coli ◽

Membrane Vesicles ◽

Whole Cells ◽

Escherichia Coli K12

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Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids

Nature Communications ◽

10.1038/s41467-021-25243-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lixia Fang ◽

Jie Fan ◽

Shulei Luo ◽

Yaru Chen ◽

Congya Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Free Fatty Acids ◽

Stress Responses ◽

Metabolic Networks ◽

Target Identification ◽

High Titer ◽

Cell Factory ◽

Microbial Cell Factory ◽

Ffa Metabolism

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

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Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Nature Communications ◽

10.1038/s41467-021-22329-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jasmine M. Hershewe ◽

Katherine F. Warfel ◽

Shaelyn M. Iyer ◽

Justin A. Peruzzi ◽

Claretta J. Sullivan ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Synthetic Biology ◽

Membrane Protein ◽

Membrane Vesicles ◽

Processing Methods ◽

Glycoprotein Synthesis ◽

On Demand ◽

Free Gene ◽

Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

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