Development of a Robust <i>Bacillus amyloliquefaciens</i> Cell Factory for Efficient Poly(γ-glutamic acid) Production from Jerusalem Artichoke

Qiu, Yibin; Zhu, Yifan; Sha, Yuanyuan; Lei, Peng; Luo, Zhengshan; Feng, Xiaohai; Li, Sha; Xu, Hong

doi:10.1021/acssuschemeng.0c02107

Cited by 29 publications

(22 citation statements)

References 45 publications

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“…In an earlier study, we found that B. amyloliquefaciens NB can efficiently use inulin to synthesize PGA and has a highly-efficient tricarboxylic acid cycle metabolic flux and glutamate synthesis ability ( Qiu et al, 2020a ; Sha et al, 2020b ). Therefore, we speculate that this strain may be optimal for synthesizing glutamate derivatives ( L -ornithine).…”

Section: Resultsmentioning

confidence: 99%

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Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin

Zhu

Hu²,

Yan³

et al. 2022

Front. Bioeng. Biotechnol.

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Bacillus amyloliquefaciens is the dominant strain used to produce γ-polyglutamic acid from inulin, a non-grain raw material. B. amyloliquefaciens has a highly efficient tricarboxylic acid cycle metabolic flux and glutamate synthesis ability. These features confer great potential for the synthesis of glutamate derivatives. However, it is challenging to efficiently convert high levels of glutamate to a particular glutamate derivative. Here, we conducted a systematic study on the biosynthesis of L-ornithine by B. amyloliquefaciens using inulin. First, the polyglutamate synthase gene pgsBCA of B. amyloliquefaciens NB was knocked out to hinder polyglutamate synthesis, resulting in the accumulation of intracellular glutamate and ATP. Second, a modular engineering strategy was applied to coordinate the degradation pathway, precursor competition pathway, and L-ornithine synthesis pathway to prompt high levels of intracellular precursor glutamate for l-ornithine synthesis. In addition, the high-efficiency L-ornithine transporter was further screened and overexpressed to reduce the feedback inhibition of L-ornithine on the synthesis pathway. Combining these strategies with further fermentation optimizations, we achieved a final L-ornithine titer of 31.3 g/L from inulin. Overall, these strategies hold great potential for strengthening microbial synthesis of high value-added products derived from glutamate.

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Section: Resultsmentioning

confidence: 99%

“…The gene knockout method refers to previous research reports ( Qiu et al, 2020b ). To knock out argF , upstream and downstream fragments of argF (Gene ID: 56457344) were amplified from B. amyloliquefaciens genome (NC_017190.1) with primer pairs argFL-F/R and argFR-F/R, respectively.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin

Zhu

Hu²,

Yan³

et al. 2022

Front. Bioeng. Biotechnol.

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“…Compared with traditional homologous recombination, CRISPR/Cas9 system gene editing requires shorter time and higher efficiency, and has been widely used in various animals, plants and microorganisms [ 38 – 40 ]. The CRISPR/Cas9 system in B. amyloliquefaciens was not reported for the first time until 2020 [ 19 ]. However, DSBs in the genome caused by Cas9 are often fatal to bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…The CRISPR-based genetic tools developed by other species and strains from the genus Bacillus can be adapted to B. amyloliquefaciens theoretically; however, it needs to be further verified through experiments due to the distinctions between different species. Qiu et al [ 19 ] reported for the first time that a dual plasmid CRISPR/Cas9n system could achieve the disruption of the B. amyloliquefaciens NB gene by integrating the Cas9n protein and sgRNA into two different vectors [ 19 ]. Zhao et al [ 20 ] further fused the nuclease-deficient mutant dCas9 of Cas9 with the ω subunit of RNA polymerase achieved gene transcription regulation in B. amyloliquefaciens 205 [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02

et al. 2022

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Background Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. Results In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. Conclusions The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.

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“…Bacteria, archaea, and eukaryotes are all γ-PGA producers, but Bacillus species are the natural, dominant, and safely produced strains [ 7 , 8 , 14 , 15 , 16 ]. Commonly, the production strains are divided into glutamic acid-dependent and glutamic acid-independent strains according to whether there is a need to add glutamate acids externally [ 17 ]. Glutamic acid dependent strains are regarded as the better producers, as adding glutamic acids can significantly increase the yield [ 1 , 9 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Microbial Synthesis of Poly-γ-Glutamic Acid: A Review

Hou

Gao

et al. 2022

Foods

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Poly-γ-glutamic acid (γ-PGA) is a natural, safe, non-immunogenic, biodegradable, and environmentally friendly glutamic biopolymer. γ-PGA has been regarded as a promising bio-based materials in the food field, medical field, even in environmental engineering field, and other industrial fields. Microbial synthesis is an economical and effective way to synthesize γ-PGA. Bacillus species are the most widely studied producing strains. γ-PGA biosynthesis involves metabolic pathway of racemization, polymerization, transfer, and catabolism. Although microbial synthesis of γ-PGA has already been used extensively, productivity and yield remain the major constraints for its industrial application. Metabolic regulation is an attempt to solve the above bottleneck problems and meet the demands of commercialization. Therefore, it is important to understand critical factors that influence γ-PGA microbial synthesis in depth. This review focuses on production strains, biosynthetic pathway, and metabolic regulation. Moreover, it systematically summarizes the functional properties, purification procedure, and industrial application of γ-PGA.

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Development of a Robust Bacillus amyloliquefaciens Cell Factory for Efficient Poly(γ-glutamic acid) Production from Jerusalem Artichoke

Cited by 29 publications

References 45 publications

Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin

Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin

Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02

Recent Advances in Microbial Synthesis of Poly-γ-Glutamic Acid: A Review

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