Cargando…
Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative
2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition an...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793955/ https://www.ncbi.nlm.nih.gov/pubmed/33425876 http://dx.doi.org/10.3389/fbioe.2020.611900 |
_version_ | 1783634106510213120 |
---|---|
author | Ni, Zhijian Li, Zhongkui Wu, Jinyong Ge, Yuanfei Liao, Yingxue Yuan, Lixia Chen, Xiangsong Yao, Jianming |
author_facet | Ni, Zhijian Li, Zhongkui Wu, Jinyong Ge, Yuanfei Liao, Yingxue Yuan, Lixia Chen, Xiangsong Yao, Jianming |
author_sort | Ni, Zhijian |
collection | PubMed |
description | 2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production. |
format | Online Article Text |
id | pubmed-7793955 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-77939552021-01-09 Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative Ni, Zhijian Li, Zhongkui Wu, Jinyong Ge, Yuanfei Liao, Yingxue Yuan, Lixia Chen, Xiangsong Yao, Jianming Front Bioeng Biotechnol Bioengineering and Biotechnology 2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production. Frontiers Media S.A. 2020-12-03 /pmc/articles/PMC7793955/ /pubmed/33425876 http://dx.doi.org/10.3389/fbioe.2020.611900 Text en Copyright © 2020 Ni, Li, Wu, Ge, Liao, Yuan, Chen and Yao. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Ni, Zhijian Li, Zhongkui Wu, Jinyong Ge, Yuanfei Liao, Yingxue Yuan, Lixia Chen, Xiangsong Yao, Jianming Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title | Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title_full | Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title_fullStr | Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title_full_unstemmed | Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title_short | Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative |
title_sort | multi-path optimization for efficient production of 2′-fucosyllactose in an engineered escherichia coli c41 (de3) derivative |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793955/ https://www.ncbi.nlm.nih.gov/pubmed/33425876 http://dx.doi.org/10.3389/fbioe.2020.611900 |
work_keys_str_mv | AT nizhijian multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT lizhongkui multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT wujinyong multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT geyuanfei multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT liaoyingxue multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT yuanlixia multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT chenxiangsong multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative AT yaojianming multipathoptimizationforefficientproductionof2fucosyllactoseinanengineeredescherichiacolic41de3derivative |