Improving the Synthesis Efficiency of Amino Acids Such as L-Lysine by Assembling Artificial Cellulosome Elements Dockerin Protein In Vivo

Li, Nan; Li, Xue; Wang, Zirui; Du, Peijun; Li, Piwu; Su, Jing; Xiao, Jing; Wang, Min; Wang, Junqing; Wang, Ruiming

doi:10.3390/fermentation8110578

Cited by 4 publications

(2 citation statements)

References 55 publications

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“…The inducible promoter Ptrc, aspC, cohA, panD and docA were cloned into pET28a (+) plasmid by selecting restriction enzyme sites EcoR I and Not I. DocA is an intracellular mutant that has undergone activity verification. Li et al (2022b) have confirmed that DocA can bind and assemble within cells, and improve product synthesis. The target fragment was cloned into a pET28a (+) plasmid.…”

Section: Construction and Transformation Of Recombinant Plasmidsmentioning

confidence: 66%

Study on the construction technology of β-alanine synthesizing Escherichia coli based on cellulosome assembly

Lü

Wang

Yang

et al. 2023

Front. Bioeng. Biotechnol.

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Introduction: β-Alanine is the only β-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of β-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method.Methods: In this study, we constructed an Escherichia coli recombinant strain for efficient β-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome.Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of β-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the β-alanine content. The key cellulosome elements, dockerin (docA) and cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. β-alanine production reached 7.439 mg/L and 25.87 mg/L in the two engineered strains. The β-alanine content reached 755.465 mg/L in a 5 L fermenter.Discussion: The content of β-alanine synthesized by constructed β-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of β-alanine using a cellulosome multi-enzyme self-assembly system.

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Section: Construction and Transformation Of Recombinant Plasmidsmentioning

confidence: 66%

Study on the construction technology of β-alanine synthesizing Escherichia coli based on cellulosome assembly

Lü

Wang

Yang

et al. 2023

Front. Bioeng. Biotechnol.

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“…After induction, the fermentation time was less than 40 h, with sampling and testing every 4 h. The concentration of bacterial cells was determined using an ultraviolet spectrophotometer (UV-6100, METASH, Shanghai, China) to detect the absorbance of the sample at 600 nm after dilution. Glucose and L-lysine contents were measured using a biosensor (BSA-90, Jinan Yanke Co., Ltd., Jinan, China) [34].…”

Section: Assembly and Detection Of Key Enzymes In L-lysine Fermentationmentioning

confidence: 99%

Improving the Synthesis Efficiency of Amino Acids by Analyzing the Key Sites of Intracellular Self-Assembly of Artificial Cellulosome

Li,

Yang,

Ren

et al. 2024

Fermentation

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To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca2+, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca2+ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca2+ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca2+ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.

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Co-localizing key pathway enzymes by protein scaffold to enhance geraniol production in Escherichia coli

Xiao,

Wang,

Zhang

et al. 2023

Industrial Crops and Products

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Improving the Synthesis Efficiency of Amino Acids Such as L-Lysine by Assembling Artificial Cellulosome Elements Dockerin Protein In Vivo

Cited by 4 publications

References 55 publications

Study on the construction technology of β-alanine synthesizing Escherichia coli based on cellulosome assembly

Study on the construction technology of β-alanine synthesizing Escherichia coli based on cellulosome assembly

Improving the Synthesis Efficiency of Amino Acids by Analyzing the Key Sites of Intracellular Self-Assembly of Artificial Cellulosome

Co-localizing key pathway enzymes by protein scaffold to enhance geraniol production in Escherichia coli

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