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Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli
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  • Dragan Miscevic,
  • Ju-Yi Mao,
  • Teshager Bitew Kefale,
  • Daryoush Abedi,
  • Murray Moo young,
  • C. Perry Chou
Dragan Miscevic
University of Waterloo
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Ju-Yi Mao
National Taiwan Ocean University
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Teshager Bitew Kefale
University of Waterloo
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Daryoush Abedi
University of Waterloo
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Murray Moo young
University of Waterloo
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C. Perry Chou
University of Waterloo
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Abstract

As issues surrounding depleting fossil fuels, climate change, and various other environmental impacts are becoming more prevalent, there is a growing interest in technological shifts toward a bio-based economy. Various advanced biotechnological tools have been developed to customize cell factories for the production of a wide range of complex fine chemicals from renewable feedstock. Herein, we report development of a microbial bioprocess for high-level and potentially economical production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis solely from glycerol (~30 g l-1) under both microaerobic and aerobic conditions, achieving up to 5.95 g l-1 (36.9% yield) and 6.93 g l-1 (50.9% yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

Peer review status:ACCEPTED

06 Mar 2020Submitted to Biotechnology and Bioengineering
09 Mar 2020Submission Checks Completed
09 Mar 2020Assigned to Editor
15 Mar 2020Reviewer(s) Assigned
17 Apr 2020Review(s) Completed, Editorial Evaluation Pending
17 Apr 2020Editorial Decision: Revise Major
01 Jun 20201st Revision Received
02 Jun 2020Assigned to Editor
02 Jun 2020Submission Checks Completed
02 Jun 2020Reviewer(s) Assigned
16 Jun 2020Review(s) Completed, Editorial Evaluation Pending
16 Jun 2020Editorial Decision: Revise Major
24 Jul 20202nd Revision Received
24 Jul 2020Submission Checks Completed
24 Jul 2020Assigned to Editor
25 Jul 2020Reviewer(s) Assigned
07 Aug 2020Editorial Decision: Revise Minor
07 Aug 2020Review(s) Completed, Editorial Evaluation Pending
19 Aug 20203rd Revision Received
19 Aug 2020Submission Checks Completed
19 Aug 2020Assigned to Editor
20 Aug 2020Reviewer(s) Assigned
26 Aug 2020Review(s) Completed, Editorial Evaluation Pending
26 Aug 2020Editorial Decision: Accept