Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system

Wang, Zhe; Dai, Yiqiang; Azi, Fidelis; Wang, Zhongjiang; Xu, Weimin; Wang, Daoying; Dong, Mingsheng; Xia, Xiudong

doi:10.1016/j.biortech.2023.130145

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…It should be pointed out that most of these efficiency prediction algorithms were developed based on experimental data of higher eukaryotes, − so the design results of microorganisms, especially prokaryotic, based on them may not be as reliable as those of higher eukaryotes. However, CHOPCHOP has also been used successfully in sgRNA design work in multiple prokaryotic species, − which demonstrates the universality of this tool at the species level. If there are more professional sgRNA design tools for microorganisms in the future, we will consider integrating them into the new version.…”

Section: Discussionmentioning

confidence: 99%

AutoESDCas: A Web-Based Tool for the Whole-Workflow Editing Sequence Design for Microbial Genome Editing Based on the CRISPR/Cas System

Yang,

Chu

et al. 2024

ACS Synth. Biol.

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Genome editing is the basis for the modification of engineered microbes. In the process of genome editing, the design of editing sequences, such as primers and sgRNA, is very important for the accurate positioning of editing sites and efficient sequence editing. The whole process of genome editing involves multiple rounds and types of editing sequence design, while the development of related whole-workflow design tools for high-throughput experimental requirements lags. Here, we propose AutoESDCas, an online tool for the end-to-end editing sequence design for microbial genome editing based on the CRISPR/Cas system. This tool facilitates all types of genetic manipulation covering diverse experimental requirements and design scenarios, enables biologists to quickly and efficiently obtain all editing sequences needed for the entire genome editing process, and empowers high-throughput strain modification. Notably, with its off-target risk assessment function for editing sequences, the usability of the design results is significantly improved. AutoESDCas is freely available at https://autoesdcas.biodesign.ac.cn/with the source code at https://github.com/tibbdc/AutoESDCas/.

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Section: Discussionmentioning

confidence: 99%

AutoESDCas: A Web-Based Tool for the Whole-Workflow Editing Sequence Design for Microbial Genome Editing Based on the CRISPR/Cas System

Yang,

Chu

et al. 2024

ACS Synth. Biol.

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“…The donor DNA was obtained through PCR amplification. To mediate the integration of the homologous arm into the chosen locus, transformation was carried out using the Cas9-recombinase-expressing plasmids pEcCas and pTarget [ 25 , 26 ]. Specific sgRNAs for the malEK and exo/cea loci were identified and ranked within the EcN genetic background using online software ( (accessed on 19 November 2023)) [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

“…The amplification conditions included an initial predenaturation step at 95 °C for 2 min, followed by a two-step reaction (95 °C, 10 s; 60 °C, 30 s) for 40 cycles. The 16S gene was chosen as the endogenous reference gene to determine the relative mRNA expression levels of zwf (glucose-6-phosphate dehydrogenase) and gnd (6-phosphogluconate dehydrogenase), applying the 2 −ΔΔCt method [ 25 ].…”

Section: Methodsmentioning

confidence: 99%

Deciphering the Crucial Roles of the Quorum-Sensing Transcription Factor SdiA in NADPH Metabolism and (S)-Equol Production in Escherichia coli Nissle 1917

Wang,

Dai,

Azi

et al. 2024

Antioxidants

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The active metabolite (S)-equol, derived from daidzein by gut microbiota, exhibits superior antioxidative activity compared with its precursor and plays a vital role in human health. As only 25% to 50% of individuals can naturally produce equol when supplied with isoflavone, we engineered probiotic E. coli Nissle 1917 (EcN) to convert dietary isoflavones into (S)-equol, thus offering a strategy to mimic the gut phenotype of natural (S)-equol producers. However, co-fermentation of EcN-eq with fecal bacteria revealed that gut microbial metabolites decreased NADPH levels, hindering (S)-equol production. Transcriptome analysis showed that the quorum-sensing (QS) transcription factor SdiA negatively regulates NADPH levels and (S)-equol biosynthesis in EcN-eq. Screening AHLs showed that SdiA binding to C10-HSL negatively regulates the pentose phosphate pathway, reducing intracellular NADPH levels in EcN-eq. Molecular docking and dynamics simulations investigated the structural disparities in complexes formed by C10-HSL with SdiA from EcN or E. coli K12. Substituting sdiA_EcN in EcN-eq with sdiA_K12 increased the intracellular NADPH/NADP+ ratio, enhancing (S)-equol production by 47%. These findings elucidate the impact of AHL-QS in the gut microbiota on EcN NADPH metabolism, offering insights for developing (S)-equol-producing EcN probiotics tailored to the gut environment.

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An efficient approach for overproduction of DNA polymerase from Pyrococcus furiosus using an optimized autoinduction system in Escherichia coli

Hadi,

Laksmi,

Helbert

et al. 2024

World J Microbiol Biotechnol

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Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system

Cited by 3 publications

References 37 publications

AutoESDCas: A Web-Based Tool for the Whole-Workflow Editing Sequence Design for Microbial Genome Editing Based on the CRISPR/Cas System

AutoESDCas: A Web-Based Tool for the Whole-Workflow Editing Sequence Design for Microbial Genome Editing Based on the CRISPR/Cas System

Deciphering the Crucial Roles of the Quorum-Sensing Transcription Factor SdiA in NADPH Metabolism and (S)-Equol Production in Escherichia coli Nissle 1917

An efficient approach for overproduction of DNA polymerase from Pyrococcus furiosus using an optimized autoinduction system in Escherichia coli

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