Journal of Food Science and Biotechnology
Abstract
[Objective ] The production of the aromatic compound salicylic acid (SA) is limited by its inhibitory effects on enzymes related to cellular energy metabolism.This study aims to develop a strategy for avoiding the impact of SA on enzymes related to cellular energy metabolism and increase the yield of SA.[Method ] A highly efficient Escherichia coli strain was designed to improve SA production by optimizing metabolic pathways and enzyme systems.[Result ] The genome-scale metabolic model GEMS 1515 was employed to predict the key bottle necks in the phosphoenolpyruvic acid (PEP) pathway and SA biosynthesis pathways.The SA yield was successfully increased from 834.82 mg/L to 1 143.56 mg/L by targeted knockout of fbaA and dcuA genes.Furthermore,the enzyme fusion strategy,combining flexible and rigid peptide linkers with TRAP scaffold proteins (TRAP 1 and TRAP 3),further improved the catalytic efficiency.The optimized strain achieved a maximum SA yield of 2 100.00 mg/L,which represented an increase of 151% over that of the initial strain.[Conclusion ] Integrating metabolic engineering,multi-enzyme cascade strategy,and machine learning demonstrate great application potential in optimizing the biosynthesis of high-value compounds.
Publication Date
12-15-2025
First Page
30
Last Page
42
DOI
10.12441/spyswjs.20241207002
Recommended Citation
WANG, Kai; PAN, Xuewei; YANG, Taowei; and RAO, Zhiming
(2025)
"Promoting the Synthesis of Salicylic Acid in Escherichia coli Based on Metabolic Engineering and Multi-enzyme Cascade Strategy,"
Journal of Food Science and Biotechnology: Vol. 44:
Iss.
12, Article 4.
DOI: 10.12441/spyswjs.20241207002
Available at:
https://spsw.spyswjs.cnjournals.com/journal/vol44/iss12/4
References
［1］ CALVEY C H，SÀNCHEZ I NOGUÉ V，WHITE A M，et al.Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering ［J］.Metabolic Engineering，2023，75：78-90.
［2］ HOMMA K，FUKUCHI S，KAWABATA T，et al.A systematic investigation identifies a significant number of probable Pseudogenes in the Escherichia coli genome［J］.Gene，2002，294（1/2）：25-33.
［3］ MISHRA A K，BAEK K H.Salicylic acid biosynthesis and metabolism：a divergent pathway for plants and bacteria［J］.Biomolecules，2021，11（5）：705.
［4］ LIN Y H，SUN X X，YUAN Q P，et al.Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli ［J］.Metabolic Engineering，2014，23：62-69.
［5］ JANDA T，SZALAI G，PÁL M.Salicylic acid signalling in plants［J］.International Journal of Molecular Sciences，2020，21（7）：2655.
［6］ ZHONG Q L，HU H L，FAN B F，et al.Biosynthesis and roles of salicylic acid in balancing stress response and growth in plants ［J］.International Journal of Molecular Sciences，2021，22（21）：11672.
［7］ WU J，ZHU W T，ZHAO Q.Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis ［J］.Journal of Integrative Plant Biology，2023，65（4）：881-887.
［8］ REKHTER D，LÜDKE D，DING Y L，et al.Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid ［J］.Science，2019，365（6452）：498-502.
［9］ TORRENS-SPENCE M P，BOBOKALONOVA A，CARBALLO V，et al.PBS 3 and EPS 1 complete salicylic acid biosynthesis from isochorismate in Arabidopsis ［J］.Molecular Plant，2019，12（12）：1577 -1586.
［10］ VARTAK R，DEORE B，SANHUEZA C A，et al.Cetuximab-based PROteolysis targeting Chimera for effectual downregulation of NSCLC with varied EGFR mutations ［J］.International Journal of Biological Macromolecules，2023，252：126413.
［11］ XIONG Y，ZHONG Y，YIM H，et al.Bridged proteolysis targeting chimera （PROTAC ） enables degradation of undruggable targets ［J］.Journal of the American Chemical Society，2022，144（49）：22622 -22632.
［12］ ZHOU J M，TRIFA Y，SILVA H，et al.NPR 1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid ［J］.Molecular Plant-Microbe Interactions，2000，13（2）：191-202.
［13］ NODA S，SHIRAI T，OYAMA S，et al.Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives ［J］.Metabolic Engineering，2016，33：119-129.
［14］ LIN Y H，SHEN X L，YUAN Q P，et al.Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin［J］.Nature Communications，2013，4：2603.
［15］ LI Z D，GAO C，YE C，et al.Systems engineering of Escherichia coli for high-level shikimate production ［J］.Metabolic Engineering，2023，75：1-11.
［16］ NOOR E，FLAMHOLZ A，BAR-EVEN A，et al.The protein cost of metabolic fluxes：prediction from enzymatic rate laws and cost minimization ［J］.PLoS Computational Biology，2016，12（11）：e1005167.
［17］ DEUTSCHER J，AKÉ F M D，DERKAOUI M，et al.The bacterial phosphoenolpyruvate：carbohydrate phosphotransferase system：regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions ［J］.Microbiology and Molecular Biology Reviews，2014，78（2）：231-256.
［18］ GERSHANOVICH V N.Phosphoenolpyruvate：carbohydrate phosphotransferase system and its role in physiology of gram-negative bacteria ［J］.Molekuliarnaia Genetika，Mikrobiologiia and Virusologiia，1998（3）：8-15.
［19］ SIEBOLD C，FLÜKIGER K，BEUTLER R，et al.Carbohydrate transporters of the bacterial phosphoenolpyruvate：sugar phosphotransferase system （PTS） ［J］.FEBS Letters，2001，504（3）：104-111.
［20］ BERENJIAN A，MAHANAMA R，TALBOT A，et al.Efficient media for high menaquinone- 7 production：response surface methodology approach ［J］.New Biotechnology，2011，28（6）：665-672.
［21］ ANDERSON K A，MADSEN A S，OLSEN C A，et al.Metabolic control by sirtuins and other enzymes that sense NAD+，NADH，or their ratio ［J］.Biochimica et Biophysica Acta （BBA）-Bioenergetics，2017，1858（12）：991-998.
［22］ SHIMIZU M.NAD+/NADH homeostasis affects metabolic adaptation to hypoxia and secondary metabolite production in filamentous fungi ［J］.Bioscience Biotechnology and Biochemistry，2018，82（2）：216-224.
［23］ GAO Y，BANG I，SEIF Y，et al.The Escherichia coli Fur pan-regulon has few conserved but many unique regulatory targets ［J］.Nucleic Acids Research，2023，51（8）：3618 -3630.
［24］ HE Z，TONEY M D.Direct detection and kinetic analysis of covalent intermediate formation in the 4-amino- 4-deoxychorismate synthase catalyzed reaction［J］.Biochemistry，2006，45（15）：5019 -5028.
［25］ BABA T，ARA T，HASEGAWA M，et al.Construction of Escherichia coli K-12 in-frame，single-gene knockout mutants：the Keio collection ［J］.Molecular Systems Biology，2006，2：2006.0008.
［26］ CHEN X Y，ZARO J L，SHEN W C.Fusion protein linkers：property，design and functionality ［J］.Advanced Drug Delivery Reviews，2013，65（10）：1357 -1369.
［27］ LEDESMA-FERNANDEZ A，VELASCO-LOZANO S，SANTIAGO-ARCOS J，et al.Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis ［J］.Nature Communications，2023，14：2587.
［28］ SPELTZ E B，NATHAN A，REGAN L.Design of protein-peptide interaction modules for assembling supramolecular structures in vivo and in vitro［J］.ACS Chemical Biology，2015，10（9）：2108 -2115.
［29］ ZHANG G Q，JOHNSTON T，QUIN M B，et al.Developing a protein scaffolding system for rapid enzyme immobilization and optimization of enzyme functions for biocatalysis ［J］.ACS Synthetic Biology，2019，8（8）：1867 -1876.
［30］ SUN L，ZHANG Q W，KONG X，et al.Highly efficient neutralizer-free L-malic acid production using engineered Saccharomyces cerevisiae ［J］.Bioresource Technology，2023，370：128580.
［31］ ZENG J，HONG Y Z，ZHAO N Q，et al.A broadly applicable，stress-mediated bacterial death pathway regulated by the phosphotransferase system （PTS） and the cAMP-Crp cascade ［J］.PNAS，2022，119（23）：e2118566119.