Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with Escherichia coli overexpressing a glucosyltransferase

Schmideder, Andreas; Priebe, Xenia; Rubenbauer, Mark; Hoffmann, Thomas; Huang, Fengting; Schwab, Wilfried; Weuster‐Botz, Dirk

doi:10.1007/s00449-016-1617-6

Cited by 17 publications

(10 citation statements)

References 22 publications

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“…In general, expression is low (≤10 mg/L; e.g., Arend, Warzecha, Hefner, & Stöckigt, 2001;Schmölzer, Gutmann, Diricks, Desmet, & Nidetzky, 2016) and poor (≤1 mg/L) in various instances (e.g., Cai et al, 2017;Welner et al, 2017). With notable exceptions (Arend et al, 2001;Dewitte et al, 2016;Priebe, Daschner, Schwab, & Weuster-Botz, 2018;Schmideder et al, 2016), the enzyme production has received relatively little attention for systematic process development. The main bottlenecks on the production efficiency thus remain largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Lemmerer

Mairhofer²,

Lepak

et al. 2019

Biotech & Bioengineering

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Sugar nucleotide‐dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology‐based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l‐arabinose‐inducible expression of the T7 phage‐derived Gp2 protein from a genome‐integrated site. The glycosyltransferase genes are encoded on conventional pET‐based expression plasmids that allow T7 RNA polymerase‐driven inducible expression by isopropyl‐β‐ d‐galactoside. Laboratory batch and scaled‐up (20 L) fed‐batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12‐fold as a result of production under growth‐decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed‐batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3‐fold) and quality (2.3‐fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth‐arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

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Section: Introductionmentioning

confidence: 99%

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Lemmerer

Mairhofer²,

Lepak

et al. 2019

Biotech & Bioengineering

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“…Thus, the table is rather intended to give an overview of existing research. Especially the comparison with results obtained by Schmideder et al (2016) is important, as there, the strain E. coli pLysS was used as the biocatalyst as well. The study does not explicitly report geranyl acetate formation.…”

Section: Discussionmentioning

confidence: 99%

Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli

et al. 2020

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Objective Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera, yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDPglucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase. Results First, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDPglucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale. Conclusion This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.

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“…To date, ILs have found applications in various types of whole-cell biocatalytic reactions, among which are biofuel production (Arai et al 2010;Ouellet et al 2011;Frederix et al 2016;Hashmi et al 2017;Pérez de los Ríos et al 2017), enantioselective reduction of ketones (Howarth et al 2001;Pfruender et al 2004Pfruender et al , 2006Lou et al 2006;Bräutigam et al 2007;Hussain et al 2007;Schroer et al 2007;Zhang et al 2008;Bräutigam et al 2009;Lou et al 2009a, b and c;Wang et al 2009Wang et al , 2013Dennewald et al 2011Dennewald et al , 2012Xiao et al 2012;Zhang et al 2012;Silva et al 2013;Zampieri et al 2013;Li et al 2015;Xu et al 2016bXu et al , 2017, enantioselective sulfoxidation (Gao et al 2014), enantioselective hydrolysis (Matsumoto et al 2014), enantioselective hydroxylation (Cornmell et al 2008;Wu et al 2011;Mao et al 2012Mao et al , 2013, dehydrogenation (He et al 2011), double-bond hydrogenation (Lenourry et al 2005;Castiglione et al 2017), glycosylation (Schmideder et al 2016), and nucleoside acylation (Yang et al 2014) (see Table 2).…”

Section: Ionic Liquids In Whole-cell Biocatalysismentioning

confidence: 99%

Ionic liquids in whole-cell biocatalysis: a compromise between toxicity and efficiency

Egorova

Ananikov

2018

Biophys Rev

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Comparison of chemical catalysis by metal complexes, enzymatic catalysis and whole-cell biocatalysis shows well-addressed advantages of the latter approach. However, a critical limitation in the practical applications originates from the high sensitivity of microorganisms to the toxic effects of organic solvents. In the present review, we consider toxic solvent properties of ionic liquid/water systems towards the development of efficient applications in practical organic transformations.

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Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with Escherichia coli overexpressing a glucosyltransferase

Cited by 17 publications

References 22 publications

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli

Ionic liquids in whole-cell biocatalysis: a compromise between toxicity and efficiency

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