An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism

Lund, Sean; Hall, Rachael; Williams, Gavin J.

doi:10.1021/acssynbio.8b00383

Cited by 87 publications

(81 citation statements)

References 56 publications

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“…Non‐canonical precursor pathways have the potential to increase terpenoid titres by overcoming inherent regulation of the MEP and MVA pathways by increasing orthogonality. Alternative pathways which use isoprenol and prenol as substrates were recently exploited for mono‐, sesqui‐, di‐ and tetraterpenoids production and showed to be a promising strategy for high‐titre terpenoid production in E. coli [13–15] . The IU pathway was compared to the MVA pathway for linalool production using the WT and the best performing bLinS variants.…”

Section: Resultsmentioning

confidence: 99%

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

et al. 2021

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Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.

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Section: Resultsmentioning

confidence: 99%

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

et al. 2021

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“…In the IUP, the substrate is the isopentenol isomer isoprenol or/and prenol, which can be directly supplemented in production media (Chatzivasileiou et al., 2019; Lund et al., 2019) or introduced via intracellular biosynthesis (Clomburg et al., 2019). One molecule of isoprenol/prenol and two molecules of ATP are consumed to produce one molecule of IPP/DMAPP (Chatzivasileiou et al., 2019).…”

Section: Biosynthesis Of Carotenoidsmentioning

confidence: 99%

Modular engineering for microbial production of carotenoids

Swofford

Sinskey

2020

Metabolic Engineering Communications

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There is an increasing demand for carotenoids due to their applications in the food, flavor, pharmaceutical and feed industries, however, the extraction and synthesis of these compounds can be expensive and technically challenging. Microbial production of carotenoids provides an attractive alternative to the negative environmental impacts and cost of chemical synthesis or direct extraction from plants. Metabolic engineering and synthetic biology approaches have been widely utilized to reconstruct and optimize pathways for carotenoid overproduction in microorganisms. This review summarizes the current advances in microbial engineering for carotenoid production and divides the carotenoid biosynthesis building blocks into four distinct metabolic modules: 1) central carbon metabolism, 2) cofactor metabolism, 3) isoprene supplement metabolism and 4) carotenoid biosynthesis. These four modules focus on redirecting carbon flux and optimizing cofactor supplements for isoprene precursors needed for carotenoid synthesis. Future perspectives are also discussed to provide insights into microbial engineering principles for overproduction of carotenoids.

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“…Thus, refining the TREE score (e.g., by weighting each factor) to better correlate in vitro and in vivo data will help identify the minimal amount of cellfree data needed to inform pathway design. Finally, our isoprenoid prototyping platform could be adapted for alternate pathway routes that utilize neryl pyrophosphate as a substrate for monoterpene synthases rather than GPP Ignea et al, 2019;Wu et al, 2019) or produce IPP and DMAPP using prenol or isoprenol as an intermediate Clomburg et al, 2019;Lund et al, 2019;Ward et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Dudley¹,

Karim²,

Nash³

et al. 2020

Preprint

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Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. Dudley, et al. -Cell-free prototyping of limonene biosynthesis 3 TOC Figure Highlights • Applied the iPROBE framework to build the nine-enzyme pathway to produce limonene • Assessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance • Iteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions • Extended pathway to biofuel precursors pinene and bisabolene Dudley, et al. -Cell-free prototyping of limonene biosynthesis 4

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An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism

Cited by 87 publications

References 56 publications

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Modular engineering for microbial production of carotenoids

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

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