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Research
Jing Wu
Jiangnan University
Corresponding Author
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In this study, we designed and in vivo reconstructed a novel four-enzyme cascade pathway for the production of D-HPG, a valuable intermediate used to produce β-lactam antibiotics and for fine-chemical synthesis, from L-tyrosine. In this pathway, we identified catalytic conversion of the substrate 4-hydroxyphenylglyoxylic acid by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg− 1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50 g/L L-tyrosine in 24 h with 92.5% conversion and > 99% ee in a 3-L fermenter, representing the highest reported D-HPG titer to date. This four-enzyme cascade provides a novel and effective enzymatic approach to industrial production of D-HPG from cheap amino acids.
Keywords
D-p-hydroxyphenylglycine, meso-diaminopimelate dehydrogenase, multi-enzyme cascade, hydride transfer distance, L-tyrosine, protein engineering
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Received 14 Mar, 2021
Reviewer #5 agreed
On 06 Mar, 2021
Reviewer #2 agreed
On 06 Mar, 2021
Reviewer #3 agreed
On 06 Mar, 2021
Reviewer #4 agreed
On 06 Mar, 2021
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Reviewer #1 agreed
On 06 Mar, 2021
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This preprint is under consideration at Bioresources and Bioprocessing. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Jing Wu
Jiangnan University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 11 Mar, 2021
No community comments so far
Editorial decision: Major revision
On 05 Apr, 2021
Review #4 received
Received 29 Mar, 2021
Review #1 received
Received 23 Mar, 2021
Review #2 received
Received 14 Mar, 2021
Review #3 received
Received 14 Mar, 2021
Reviewer #5 agreed
On 06 Mar, 2021
Reviewer #2 agreed
On 06 Mar, 2021
Reviewer #3 agreed
On 06 Mar, 2021
Reviewer #4 agreed
On 06 Mar, 2021
Reviewers invited
Invitations sent on 06 Mar, 2021
Reviewer #1 agreed
On 06 Mar, 2021
Editor assigned
On 05 Mar, 2021
Editor invited
On 05 Mar, 2021
Submission checks complete
On 04 Mar, 2021
First submitted
On 02 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In this study, we designed and in vivo reconstructed a novel four-enzyme cascade pathway for the production of D-HPG, a valuable intermediate used to produce β-lactam antibiotics and for fine-chemical synthesis, from L-tyrosine. In this pathway, we identified catalytic conversion of the substrate 4-hydroxyphenylglyoxylic acid by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg− 1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50 g/L L-tyrosine in 24 h with 92.5% conversion and > 99% ee in a 3-L fermenter, representing the highest reported D-HPG titer to date. This four-enzyme cascade provides a novel and effective enzymatic approach to industrial production of D-HPG from cheap amino acids.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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