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Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP
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  • Qingyan Yuan,
  • Minhui Wu,
  • Yibo Liao,
  • Shuli Liang,
  • Yuan Lu,
  • Ying Lin
Qingyan Yuan
South China University of Technology

Corresponding Author:yuan.qy@mail.scut.edu.cn

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Minhui Wu
South China University of Technology
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Yibo Liao
South China University of Technology
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Shuli Liang
South China University of Technology
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Yuan Lu
Tsinghua University
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Ying Lin
South China University of Technology
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Abstract

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a novel strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split GFP. This strategy featured two main advantages: 1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and 2) the expression level and activity of each homolog was determined [simultaneously](javascript:;) by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.
29 Oct 2022Submitted to Biotechnology and Bioengineering
29 Oct 2022Submission Checks Completed
29 Oct 2022Assigned to Editor
29 Oct 2022Review(s) Completed, Editorial Evaluation Pending
31 Oct 2022Reviewer(s) Assigned
17 Nov 2022Editorial Decision: Revise Major
15 Dec 20221st Revision Received
19 Dec 2022Submission Checks Completed
19 Dec 2022Assigned to Editor
19 Dec 2022Review(s) Completed, Editorial Evaluation Pending
19 Dec 2022Reviewer(s) Assigned
30 Dec 2022Editorial Decision: Accept