Guiding efficient microbial synthesis of non-natural chemicals by physicochemical properties of reactants

Willrodt, Christian; Karande, Rohan; Schmid, Andreas; Julsing, Mattijs K.

doi:10.1016/j.copbio.2015.03.010

Cited by 26 publications

(25 citation statements)

References 66 publications

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“…An integrated approach on the combination of a powerful catalyst with the specific reaction and reactor design addressing all issues critical for high productivity (as described in a recent review by Willrodt et al, 2015), opens new opportunities for cyclohexanol process development. CHX100.…”

Section: Resultsmentioning

confidence: 99%

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms

Karande

Debor

Salamanca

et al. 2015

Biotech & Bioengineering

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The applications of biocatalysts in chemical industries are characterized by activity, selectivity, and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g L tube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile, and have low solubility in water.

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Section: Resultsmentioning

confidence: 99%

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms

Karande

Debor

Salamanca

et al. 2015

Biotech & Bioengineering

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“…This allows for the exploitation of microorganisms to function as "microfactories" to produce specific compounds that are of high market value in various industrial sectors (12). However, in order for a strain to be viable for industrial scale-up, the engineered strain must be able to achieve high yields, high production rates, and high final titers of the desired compounds (13).…”

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confidence: 99%

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

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bAnthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (؉)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

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“…These compartments are highly hydrophobic due to the presence of oily substances. The use of a two-liquid phase setup might mimic these natural conditions (Willrodt et al, 2015). The low aqueous solubility of CBGA (0.002152 mg L −1 , (http://www.chemspider.com/ChemicalStructure.24784964.html, 2015)) led to mass transfer limitations for the substrate in the single aqueous phase activity assay (substrate availability).…”

Section: Process Efficiency Depends On Properties Of the Enzyme Reacmentioning

confidence: 99%

“…In Nature, specialized organisms developed solutions to enable chemical synthesis, e.g., by the synthesis of specific enzymes and specified reaction compartments. The introduction of enzymes, but also the translation of other natural solutions in technical applications might support chemical synthesis (Gröger and Asano, 2011;Willrodt et al, 2015). The successful implementation of biocatalytic processes for chemical synthesis is determined by the availability of an specific biocatalyst (Schmid et al, 2001) and its kinetic characteristics, but also the physico-chemical properties of the reactants, the production technology ( Fig.…”

Section: Introductionmentioning

confidence: 98%

Δ9-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids

Länge

Schmid

Julsing

2015

Journal of Biotechnology

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Guiding efficient microbial synthesis of non-natural chemicals by physicochemical properties of reactants

Cited by 26 publications

References 66 publications

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Δ9-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids

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