High-level extracellular expression of phospholipase D by combinatorial fine-tuning strategy in Bacillus subtilis for efficient biosynthesis of phosphatidic acid

Peng Zhang; Xin-An Mao; Jin-Song Gong; Xiao-Li Kong; Chang Su; Xiao-Mei Zhang; Zhi-Ming Rao; Zheng-Hong Xu; Jin-Song Shi

doi:10.1016/j.biortech.2024.131492

High-level extracellular expression of phospholipase D by combinatorial fine-tuning strategy in Bacillus subtilis for efficient biosynthesis of phosphatidic acid

Bioresour Technol. 2024 Dec:414:131492. doi: 10.1016/j.biortech.2024.131492. Epub 2024 Sep 27.

Authors

Peng Zhang¹, Xin-An Mao², Jin-Song Gong³, Xiao-Li Kong⁴, Chang Su⁵, Xiao-Mei Zhang⁶, Zhi-Ming Rao⁷, Zheng-Hong Xu⁸, Jin-Song Shi⁹

Affiliations

¹ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China. Electronic address: [email protected].
² Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: [email protected].
³ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: [email protected].
⁴ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: [email protected].
⁵ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: [email protected].
⁶ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: [email protected].
⁷ Institute of Future Food Technology, JITRI, Yixing 214200, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China. Electronic address: [email protected].
⁸ Institute of Future Food Technology, JITRI, Yixing 214200, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China; College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, PR China. Electronic address: [email protected].
⁹ Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, PR China; Institute of Future Food Technology, JITRI, Yixing 214200, PR China. Electronic address: [email protected].

PMID: 39343177
DOI: 10.1016/j.biortech.2024.131492

Abstract

Although Bacillus subtilis shows promise as a potential microbial cell factory for phospholipase D (PLD) expression, its production capacity remains insufficient. In this study, a secretory expression system, by co-optimization the promoter and signal peptides and employing a fed-batch fermentation strategy, was constructed to enhance expression of PLD from separate sources. The highest PLD production of 4056.9 U/mL was observed using this system, with a PLD production efficiency of 52.0 U/mL/h. Finally, a phosphatidic acid (PA) biosynthesis system was established using the constructed PLD as a catalyst, which achieved a PA yield of 219.1 g/L. This is the highest PLD production and PA yield reported globally to date. The protocol has significant potential for application for industrial PLD production as well as enzymatic phospholipids modification and also provides a valuable reference for overexpressing proteins in B. subtilis.

Keywords: Bacillus subtilis; Phosphatidic acid; Phospholipase D; Secretion expression.

MeSH terms

Bacillus subtilis* / enzymology
Bacillus subtilis* / genetics
Bacillus subtilis* / metabolism
Extracellular Space
Fermentation
Phosphatidic Acids* / biosynthesis
Phosphatidic Acids* / metabolism
Phospholipase D* / metabolism
Promoter Regions, Genetic
Protein Sorting Signals

Substances

Phospholipase D
Phosphatidic Acids
Protein Sorting Signals