Alternative metabolic pathways and strategies to high-titre terpenoid production in<i>Escherichia coli</i>

Rinaldi, Mauro A.; Ferraz, Clara A.; Scrutton, Nigel S.

doi:10.1039/d1np00025j

Cited by 49 publications

(39 citation statements)

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“…Prior studies have suggested that instability in titers of terpenoids and other natural products may be due to plasmid recombination events, which in turn reduces in vivo production of toxic intermediates and products generated by full length constructs ( Rinaldi et al, 2021 ). This may be especially relevant for pathways where either large or multiple plasmids need to be maintained.…”

Section: Resultsmentioning

confidence: 99%

“…The results indicated that linalool significantly affects cell growth rate at levels < 1 g/L, with high concentrations (20 g/L) leading to a rapid loss of cell density after the addition of linalool ( Supplementary Figure S6 ). Therefore, we performed Adaptive Laboratory Evolution (ALE) to increase the tolerance of E. coli DH5α towards linalool by performing continuous cultivation in increasing concentrations of linalool ( Rinaldi et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…Production of these often-high value chemicals through the engineering of biology has been studied extensively, with each final product derived from common precursor pathways, such as the mevalonate (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP) precursor upregulation pathway(s) ( Martin et al, 2003 ). Enantiopure monoterpenoids can be produced to g/L levels using Escherichia coli as the microbial host ( Rinaldi et al, 2021 ), which is often higher than using other microorganisms ( Moser and Pichler, 2019 ). One high value monoterpenoid is the natural product ( R )-linalool, an odoriferous acyclic compound widely used as a scent in cosmetic and household cleaning products ( Ferraz et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Bioproduction of Linalool From Paper Mill Waste

Rinaldi

Tait

Toogood

et al. 2022

Front. Bioeng. Biotechnol.

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A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A “cookie-cutter” approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both ‘hit and miss’ outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Bioproduction of Linalool From Paper Mill Waste

Rinaldi

Tait

Toogood

et al. 2022

Front. Bioeng. Biotechnol.

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“…The use of enzymes from both natural sources or produced in recombinant form, as raw or highly purified extracts, is also becoming increasingly common [ 7 , 8 , 9 ]. Enzymes can be used in cascades that mimic synthetic biochemical pathways, or they can replace some steps in the total synthesis of natural and non-natural molecules with high structural complexity [ 10 , 11 , 12 , 13 , 14 ]. However, the use of enzymes can be challenging on a large scale due to their high production costs, low operational stability, and difficulties in recovering them from the reaction mixtures [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications

Fabio

Iazzetti

Incocciati

et al. 2022

IJMS

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Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes the oxidative deamination of a wide range of primary amines into the corresponding aldehydes but its use as a biocatalyst is challenging due to the possible inactivation that might occur at high product concentrations. Here, we show that the enzyme’s performance can be greatly improved by immobilization on solid supports. The best results are achieved using amino-functionalized magnetic microparticles: the immobilized enzyme retains its activity, greatly improves its thermostability (4 h at 75 °C), and can be recycled up to 8 times with a set of aromatic ethylamines. After the last reaction cycle, the overall conversion is about 90% for all tested substrates, with an aldehyde production ranging between 100 and 270 mg depending on the substrate used. As a proof concept, one of the aldehydes thus produced was successfully used for the biomimetic synthesis of a non-natural benzylisoquinoline alkaloid.

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“…Alternative pathways, including additional entry points into the MVA and MEP pathways, archaeal MVA pathways, and artificial pathways have also been identified at the sequence level. [6] Prenyl pyrophosphate (prenyl‐PP) synthases catalyse the head‐to‐tail condensation of IPP and DMAPP into prenyl‐PP substrates of varying lengths such as geranyl pyrophosphate (GPP, C 10 ), farnesyl pyrophosphate (FPP, C 15 ), geranylgeranyl pyrophosphate (GGPP, C 20 ) and geranyl‐farnesyl pyrophosphate (GFPP, C 25 ), the pre‐cursors for mono‐, sesqui‐, di‐ and sesterterpenoids, respectively. These prenyl‐PP substrates are converted into structurally diverse terpene scaffolds by terpene synthases (TSs).…”

Section: Introductionmentioning

confidence: 99%

Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

Leferink

Scrutton

2021

ChemBioChem

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Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence‐function relationship within the TS family, making product‐outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high‐throughput screening, and machine‐learning that will allow more predictive engineering of these fascinating enzymes in the near future.

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Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

Abstract: Terpenoids are a diverse group of chemicals used in a wide range of industries. They can be made through alternative metabolic pathways to reach the higher needed for biomanufacturing.

Cited by 49 publications

References 325 publications

Bioproduction of Linalool From Paper Mill Waste

Bioproduction of Linalool From Paper Mill Waste

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications

Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

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