Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid in<i>Bacillus amyloliquefaciens</i>LL3

Gao, Weixia; Liu, Fenghong; Zhang, Wei; Quan, Yufen; Dang, Yulei; Feng, Jun; Gu, Yanyan; Wang, Shufang; Song, Chunyan; Yang, Chao

doi:10.1002/mbo3.398

Cited by 24 publications

(21 citation statements)

References 30 publications

(51 reference statements)

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“…For example, the synthesis of extracellular polysaccharide and lipopolysaccharide in Bacillus amyloliquefaciens LL3 was blocked to decrease competitive byproduct production to improve γ‐PGA production (Feng et al, ). The deletion of genes itu , bae , srf , and fen encoding for antibiotic substances increased the γ‐PGA yield by 35% (Gao et al, ). Deletion of the genes encoding the γ‐PGA hydrolase, pgdS , and cwlO , also enhanced γ‐PGA yield (Scoffone et al, ).…”

Section: Introductionmentioning

confidence: 99%

Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

Cai

Chen

et al. 2018

Biotech & Bioengineering

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Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

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Section: Introductionmentioning

confidence: 99%

Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

Cai

Chen

et al. 2018

Biotech & Bioengineering

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“…In our previous work, we found that in‐frame deletions of itu , srf and fen gene clusters encoding antibiotic substances could reduce the culture viscosity of B. amyloliquefaciens (Gao et al ., ). In combination with the above results, we carried out in‐frame deletion of srf and itu gene clusters, whose size was 26.16 Kbp and 37.24 Kbp, both in NK‐A7 and NK‐A9E.…”

Section: Resultsmentioning

confidence: 97%

“…To construct the gene deletion vectors, the temperature‐sensitive pKSU plasmid with a upp expression cassette and primers N‐UP‐F/R, N‐DN‐F/R (N represents relevant gene name) were used. The deletion plasmids were constructed as previously reported (Gao et al ., ).…”

Section: Methodsmentioning

confidence: 97%

Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

Gao

He²,

Zhang

et al. 2019

Microbial Biotechnology

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Summary Poly‐γ‐glutamic acid (γ‐PGA) is a biocompatible and biodegradable polypeptide with wide‐ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ‐PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ‐PGA production by LL3. In this study, the γ‐PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism‐relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK‐A6 synthesized 4.84 g l−1 γ‐PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter PC2up was inserted into the upstream of icd gene, to generate strain NK‐A7, which further led to a 33.5% improvement in the γ‐PGA titre, achieving 6.46 g l−1. The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK‐A9E, which showed a comparable γ‐PGA titre with strain NK‐A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK‐A7 and NK‐A9E. The resulting strain NK‐A11 exhibited the highest γ‐PGA titre (7.53 g l−1), with a 2.05‐fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ‐PGA production in B. amyloliquefaciens fermentation.

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“…RNA-seq analyses of LL3 were carried out according to our previous methods [32]. The expression levels of the predicted genes were quanti ed as the FPKM value [43]. The upstream regions of genes with different FPKM values were submitted online (http://www.fruit y.org/seq_tools/promoter) for promoter prediction.…”

Section: Rna-seq Promoter Prediction and Construction Of Reporter Gmentioning

confidence: 99%

“…Prior to HPLC-MS analysis, the supernatant was concentrated through a vacuum rotary evaporator and ltered via a 0.22-μm lter. Surfactin was analyzed and quanti ed by HPLC-MS equipped with a C18 column (Innoval ODS-2, 250 mm × 4.6 mm × 5 μm, Phenomenex, USA) using a validated method described previously [17,43]. The extracted surfactin samples (20 μL) were injected into the HPLC-MS system with a mobile phase consisting of acetonitrile and water (55:45, v/v) at a ow rate of 0.8 mL/min.…”

Section: Surfactin Isolation and Hplc-ms Analysesmentioning

confidence: 99%

A combination of genome reduction and promoter engineering can enhance surfactin production by Bacillus amyloliquefaciens LL3

Zhang

Huo

Song

et al. 2020

Preprint

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Background: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis.Results: In this study, a genome-reduced strain GR167 lacking ~4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 harbored faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the promising chassis GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167.Conclusions: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a promising chassis for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

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Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid inBacillus amyloliquefaciensLL3

Cited by 24 publications

References 30 publications

Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

A combination of genome reduction and promoter engineering can enhance surfactin production by Bacillus amyloliquefaciens LL3

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