Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase
Abstract Background In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) ...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-11-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-023-02249-1 |
| _version_ | 1797413618682167296 |
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| author | Sabine Schelch Manuel Eibinger Jasmin Zuson Jürgen Kuballa Bernd Nidetzky |
| author_facet | Sabine Schelch Manuel Eibinger Jasmin Zuson Jürgen Kuballa Bernd Nidetzky |
| author_sort | Sabine Schelch |
| collection | DOAJ |
| description | Abstract Background In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3′-sialyllactose (3SL). Results Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5′-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. Conclusions Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production. |
| first_indexed | 2024-03-09T05:20:35Z |
| format | Article |
| id | doaj.art-d72c0531657a48b3a431d6a016204d07 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-09T05:20:35Z |
| publishDate | 2023-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-d72c0531657a48b3a431d6a016204d072023-12-03T12:41:02ZengBMCMicrobial Cell Factories1475-28592023-11-0122111510.1186/s12934-023-02249-1Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferaseSabine Schelch0Manuel Eibinger1Jasmin Zuson2Jürgen Kuballa3Bernd Nidetzky4Austrian Centre of Industrial BiotechnologyInstitute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI GrazAustrian Centre of Industrial BiotechnologyGALAB Laboratories GmbHAustrian Centre of Industrial BiotechnologyAbstract Background In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3′-sialyllactose (3SL). Results Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5′-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. Conclusions Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.https://doi.org/10.1186/s12934-023-02249-1Sialo-oligosaccharides3ʹ-Sialyllactoseα2,3‐SialyltransferaseWhole-cell bio-catalysisMultienzyme cascade reactionCo-expression |
| spellingShingle | Sabine Schelch Manuel Eibinger Jasmin Zuson Jürgen Kuballa Bernd Nidetzky Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase Microbial Cell Factories Sialo-oligosaccharides 3ʹ-Sialyllactose α2,3‐Sialyltransferase Whole-cell bio-catalysis Multienzyme cascade reaction Co-expression |
| title | Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase |
| title_full | Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase |
| title_fullStr | Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase |
| title_full_unstemmed | Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase |
| title_short | Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase |
| title_sort | modular bioengineering of whole cell catalysis for sialo oligosaccharide production coordinated co expression of cmp sialic acid synthetase and sialyltransferase |
| topic | Sialo-oligosaccharides 3ʹ-Sialyllactose α2,3‐Sialyltransferase Whole-cell bio-catalysis Multienzyme cascade reaction Co-expression |
| url | https://doi.org/10.1186/s12934-023-02249-1 |
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