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Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli
Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cof...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463868/ https://www.ncbi.nlm.nih.gov/pubmed/32781536 http://dx.doi.org/10.3390/molecules25163567 |
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author | Li, Zhongkui Ni, Zhijian Chen, Xiangsong Wang, Gang Wu, Jinyong Yao, Jianming |
author_facet | Li, Zhongkui Ni, Zhijian Chen, Xiangsong Wang, Gang Wu, Jinyong Yao, Jianming |
author_sort | Li, Zhongkui |
collection | PubMed |
description | Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL. |
format | Online Article Text |
id | pubmed-7463868 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-74638682020-09-04 Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli Li, Zhongkui Ni, Zhijian Chen, Xiangsong Wang, Gang Wu, Jinyong Yao, Jianming Molecules Article Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL. MDPI 2020-08-06 /pmc/articles/PMC7463868/ /pubmed/32781536 http://dx.doi.org/10.3390/molecules25163567 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Li, Zhongkui Ni, Zhijian Chen, Xiangsong Wang, Gang Wu, Jinyong Yao, Jianming Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title | Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title_full | Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title_fullStr | Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title_full_unstemmed | Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title_short | Multi-Enzymatic Cascade One-Pot Biosynthesis of 3′-Sialyllactose Using Engineered Escherichia coli |
title_sort | multi-enzymatic cascade one-pot biosynthesis of 3′-sialyllactose using engineered escherichia coli |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463868/ https://www.ncbi.nlm.nih.gov/pubmed/32781536 http://dx.doi.org/10.3390/molecules25163567 |
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