Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development

Schmölzer, Katharina; Gutmann, Alexander; Diricks, Margo; Desmet, Tom; Nidetzky, Bernd

doi:10.1016/j.biotechadv.2015.11.003

Cited by 169 publications

(149 citation statements)

References 174 publications

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“…B) showed a predominant band at an apparent molecular mass of >80 kDa, representing the SuSyAc subunit. The native molecular mass of SuSyAc estimated by SEC was about 367 kDa, consistent with a SuSy homotetramer as reported for SuSys from other sources (Schmölzer et al, ). Under the conditions described, the target expression level of SuSyAc was clearly reached.…”

Section: Introductionsupporting

confidence: 88%

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Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

Schmölzer

Lemmerer

Gutmann

et al. 2016

Biotech & Bioengineering

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Nucleotide sugar‐dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine‐5′‐diphosphate glucose (UDP‐glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole‐cell biotransformation. The UDP‐glc production had excellent performance metrics of ∼100 gproduct/L, 86% yield (based on UDP), and a total turnover number of 103 gUDP‐glc/gcell dry weight at a space‐time yield of 10 g/L/h. Using efficient chromatography‐free DSP, the UDP‐glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. © 2016 Wiley Periodicals, Inc.

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Section: Introductionsupporting

confidence: 88%

“…Under the conditions described, the target expression level of SuSyAc was clearly reached. Furthermore, the SuSy yield was 10‐ to 100‐fold increased compared to maximum enzyme titers of different SuSys in the literature (3 and 25 mg/L medium , Schmölzer et al, ).…”

Section: Introductionmentioning

confidence: 69%

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

Schmölzer

Lemmerer

Gutmann

et al. 2016

Biotech & Bioengineering

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“…It is a functional homotetramer composed of 92.2 kDa subunits. GmSuSy has previously been used for UDP‐glucose recycling in glycosylation reactions by coupled GTs, including UGT71A15 (Scheme ; Lepak et al, ; Schmölzer et al, ; Schmölzer et al, ).…”

Section: Resultsmentioning

confidence: 99%

“…Escherichia coli is most often used. 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.…”

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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“…Lipoxygenases, a nonheme iron-containing group of dioxygenases involved in vegetative growth, defense responses and anti-microbial actions are enhanced in their abundance. In contrast, sucrose synthases 1 and 2, enzymes involved in the breakdown of the disaccharide sucrose, are down-regulated upon exposer to BL (Porta and Rocha-Sosa 2002, Kutschera and Niklas 2013, Schmolzer et al 2016). These data document that lipid- and sucrose metabolism are both rapidly affected by BL.…”

Section: Discussionmentioning

confidence: 99%

Seedling development in maize cv. B73 and blue light-mediated proteomic changes in the tip vs. stem of the coleoptile

2016

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In 2009, the draft genome of the reference inbred line of maize (Zea mays L. spp. mays cv. B73) was published so that, using this specific corn variety, molecular analyses of physiological processes became possible. However, the morphology and developmental patterns of B73 maize, compared with that of the more frequently used hybrid varieties, have not yet been analyzed. Here, we describe organ development in seedlings of B73 maize and in those of 6 other hybrid cultivars, and document significant morphological as well as quantitative differences between these varieties of Z. mays. In a second set of experiments we used etiolated seedlings of B73 maize to analyze the effect of blue light (BL) on the patterns of proteins in the tip vs. growing region of this sheath-like organ. By using two-dimensional difference gel electrophoresis (2-D DIGE), coupled with tandem mass spectrometry, we detected, in the microsomal fraction of maize coleoptile tips, rapid changes in the abundance of protein spots of maize phototropin 1 and several metabolic enzymes. In the sub-apical (growing) region of the coleoptile, proteomic changes were less pronounced. These results suggest that the tip of the coleoptile of B73 maize may serve as a unique model system for dissecting BL-responses in a light-sensitive plant organ of known function.

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Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development

Cited by 169 publications

References 174 publications

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

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

Seedling development in maize cv. B73 and blue light-mediated proteomic changes in the tip vs. stem of the coleoptile

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