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Protein engineering, cofactor engineering and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A
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  • Haoyu Liu,
  • Wanqing Wei,
  • Wei Song,
  • Simin Gu,
  • Cong Gao,
  • Xiulai Chen,
  • Jia Liu,
  • Liang Guo,
  • Jing Wu,
  • Liming Liu
Haoyu Liu
State Key Laboratory of Food Science and Technology
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Wanqing Wei
State Key Laboratory of Food Science and Technology
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Wei Song
Jiangnan University
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Simin Gu
State Key Laboratory of Food Science and Technology
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Cong Gao
State Key Laboratory of Food Science and Technology
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Xiulai Chen
State Key Laboratory of Food Science and Technology
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Jia Liu
State Key Laboratory of Food Science and Technology
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Liang Guo
State Key Laboratory of Food Science and Technology
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Jing Wu
Jiangnan University
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Liming Liu
State Key Laboratory of Food Science and Technology

Corresponding Author:mingll@jiangnan.edu.cn

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Abstract

Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole-cell catalytic production of CSA. Using mechanism-based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its T m and half-life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1-fold. Via cofactor engineering, we designed a dual cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole-cell catalytic system of CSA production with a 89.5% conversion rate. This whole-cell catalytic process provides a promising method for the industrial production of CSA.
06 Jan 2023Submitted to Biotechnology and Bioengineering
09 Jan 2023Submission Checks Completed
09 Jan 2023Assigned to Editor
09 Jan 2023Review(s) Completed, Editorial Evaluation Pending
15 Jan 2023Reviewer(s) Assigned
11 Feb 2023Editorial Decision: Revise Major
27 Feb 20231st Revision Received
27 Feb 2023Submission Checks Completed
27 Feb 2023Assigned to Editor
27 Feb 2023Review(s) Completed, Editorial Evaluation Pending
05 Mar 2023Reviewer(s) Assigned
26 Mar 2023Editorial Decision: Revise Major
28 Mar 20232nd Revision Received
31 Mar 2023Submission Checks Completed
31 Mar 2023Assigned to Editor
31 Mar 2023Review(s) Completed, Editorial Evaluation Pending
02 Apr 2023Editorial Decision: Accept