Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.
Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc.
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China.
Xiaomei
X
Lv
College of Life Sciences, Huzhou University, Huzhou, PR China.
Jiali
J
Gu
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China.
Fan
F
Wang
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China.
Wenping
W
Xie
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China.
Min
M
Liu
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China. yelidan@zju.edu.cn.
Lidan
L
Ye
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, 310027, Hangzhou, PR China. yuhongwei@zju.edu.cn.
Hongwei
H
Yu
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D002070:Butadienes; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D045782:Hemiterpenes / Q000096:biosynthesis* / Q000235:genetics; D008657:Metabolic Clearance Rate; D060847:Metabolic Engineering / Q000379:methods*; D010420:Pentanes; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002070:Butadienes; D045782:Hemiterpenes; D010420:Pentanes; D011994:Recombinant Proteins; C005059:isoprene
MEP pathway; directed co-evolution; isoprene; modular pathway engineering