Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Zhu, Jiangfeng; Cai, Dongbo; Xu, Haixia; Liu, Ziwei; Zhang, Bowen; Wu, Fei; Li, Junhui; Chen, Shouwen

doi:10.1016/j.synbio.2018.10.009

Cited by 25 publications

(24 citation statements)

References 40 publications

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“…Here, Leu, derived from pyruvate, acted as the initial precursor for pulcherriminic acid synthesis. Because of the low concentration of intracellular Leu in B. licheniformis DW2 (27), Leu supply might be a bottleneck for pulcherriminic acid production. Vogt et al (28) have obtained an efficient L-Leu production strain of Corynebacterium glutamicum by removing the feedback resistance and deleting the repressor LtbR for L-Leu synthesis, and Leu yield was increased to 24 g/liter under optimized fed-batch fermentation.…”

Section: Discussionmentioning

confidence: 99%

“…Vogt et al (28) have obtained an efficient L-Leu production strain of Corynebacterium glutamicum by removing the feedback resistance and deleting the repressor LtbR for L-Leu synthesis, and Leu yield was increased to 24 g/liter under optimized fed-batch fermentation. In addition, Zhu et al (27) improved the accumulation of intracellular branched-chain amino acids (BCAAs) by overexpressing the BCAA importer BrnQ, which led to a 22.4% increase in the bacitracin yield. In our work, the concentration of intracellular Leu was increased by 227.6% via overexpressing ilvBHC-leuABCD and deleting bkdAB, and a 49.0% increase of pulcherriminic acid yield was achieved.…”

Section: Discussionmentioning

confidence: 99%

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Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production

Wang

Zhang

et al. 2020

Appl Environ Microbiol

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The cyclodipeptide pulcherriminic acid, produced by Bacillus licheniformis, is derived from cyclo(l-Leu–l-Leu) and possesses excellent antibacterial activities. In this study, we achieved the high-level production of pulcherriminic acid via multistep metabolic engineering of B. licheniformis DWc9n*. First, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD, involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene bkdAB, encoding a branched-chain α-keto acid dehydrogenase in W1. As a result, the intracellular Leu content and pulcherriminic acid yield of W2 reached 147.4 mg/g DCW (dry cell weight) and 189.9 mg/liter, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Second, strain W3 was constructed through overexpressing the leucyl-tRNA synthase gene leuS in W2, and it produced 367.7 mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster yvmC-cypX in W3 was replaced with a proven strong promoter, PbacA, to produce the strain W4, and its pulcherriminic acid yield was increased to 507.4 mg/liter. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene yvmA in W4, resulting in the W4/pHY-yvmA strain, which yielded 556.1 mg/liter pulcherriminic acid, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to the high-level production of other cyclodipeptides. IMPORTANCE Pulcherriminic acid is a cyclodipeptide derived from cyclo(l-Leu–l-Leu), which shares the same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be the perfect candidates for antibacterial and anti-plant-pathogenic fungal agents. In this study, we obtained the promising W4/pHY-yvmA pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY-yvmA strain is able to achieve 556.1 mg/liter pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production

Wang

Zhang

et al. 2020

Appl Environ Microbiol

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“…Since soybean meal contains Met , overexpression of MetP improved the accumulation of intracellular Met, which further benefit bacitracin production. Meantime, several other amino acid transporters (YdhG, BrnQ, and LysE) have also been engineered for bacitracin production (Li et al, 2018;Zhu et al, 2018;Wu et al, 2019), indicated that engineering amino acid transporter was an efficient strategy for bacitraicn production. Similar to Met transportation, several other amino acids also have multiple corresponding transporters.…”

Section: Discussionmentioning

confidence: 99%

“…The synthetic pathways of Lysine (Lys) and Ornithine (Orn) were strengthened, which led to 28.95 and 16.5% increases of bacitracin yields, respectively (Wu et al, 2019;Yu et al, 2019). BCAA transporters BrnQ and YdhG were engineered to improve intracellular BCAA accumulations for bacitraicn synthesis (Li et al, 2018;Zhu et al, 2018). Additionally, the main regulators in carbon, nitrogen and phosphorus metabolisms were engineered, which led to a 35.72% increase of bacitraicn yield (Cai et al, 2019b).…”

Section: Introductionmentioning

confidence: 99%

Enhanced Bacitracin Production by Systematically Engineering S-Adenosylmethionine Supply Modules in Bacillus licheniformis

Cai

Zhang

Zhu

et al. 2020

Front. Bioeng. Biotechnol.

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Bacitracin is a broad-spectrum veterinary antibiotic that widely used in the fields of veterinary drug and feed additive. S-Adenosylmethionine (SAM) is a critical factor involved in many biochemical reactions, especially antibiotic production. However, whether SAM affects bacitracin synthesis is still unknown. Here, we want to analyze the relationship between SAM supply and bacitracin synthesis, and then metabolic engineering of SAM synthetic pathway for bacitracin production in Bacillus licheniformis. Firstly, our results implied that SAM exogenous addition benefited bacitracin production, which yield was increased by 12.13% under the condition of 40 mg/L SAM addition. Then, SAM synthetases and Methionine (Met) synthetases from B. licheniformis, Corynebacterium glutamicum, and Saccharomyces cerevisiae were screened and overexpressed to improve SAM accumulation, and the combination of SAM synthetase from S. cerevisiae and Met synthetase from B. licheniformis showed the best performance, and 70.12% increase of intracellular SAM concentration (31.54 mg/L) and 13.08% increase of bacitraicn yield (839.54 U/mL) were achieved in resultant strain DW2-KE. Furthermore, Met transporters MetN and MetP were, respectively, identified as Met exporter and importer, and bacitracin yield was further increased by 5.94% to 889.42 U/mL via deleting metN and overexpressing metP in DW2-KE, attaining strain DW2-KENP. Finally, SAM nucleosidase gene mtnN and SAM decarboxylase gene speD were deleted to block SAM degradation pathways, and bacitracin yield of resultant strain DW2-KENPND reached 957.53 U/mL, increased by 28.97% compared to DW2. Collectively, this study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.

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“…Amino acids, especially BCAAs, are often used in the antibiotics production. It was reported that enhancement of precursor amino acid supplies improved bacitracin production by activation of branched chain amino acid transporter BrnQand deletion of its regulator gene lrp in Bacillus licheniformis [36]. BCAAs were used in improving secondary metabolites of Streptomyces spp, for example bitespiramycin [37].…”

Section: Discussionmentioning

confidence: 99%

Transcriptome analysis of the regulation of natamycin biosynthesis in Streptomyces natalensis HW-2 by fungal elicitor

Wang

Shen

Yuan

et al. 2019

Preprint

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Background Natamycin is a polyene macrolide polyketide antibiotics and used in 150 countries as a natural food preservative. Streptomyces natalensis is an important producer. Elicitation had been approved to be an effective method to improve the biosynthesis of secondary metabolites. Fungal elicitor from Penicillium chrysogenum AS 3.5163 showed inductive effect on the biosynthesis of natamycin in S. natalensis HW-2 fermentation. However, regarding the global gene expression of natamycin in response to fungal elicitor is not still reported. Results RNA-Seq analysis showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. The fungal elicitor had stronger effects on the transcription level of S. natalensis HW-2 at 80 h than that at 40 h. Gene Ontology (GO) enrichment analysis of DEGs showed significant enrichment in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the fungal elicitor mainly affected the expression levels of some genes about cellular process, metabolism and genetic information, especially in pentose phosphate pathway (PPP), glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA). KEGG pathway showed that fungal elicitor had a greater influence on the metabolism of branched-chain amino acids (BCAAs). Among them, 23 DEGs associated with BCAAs metabolism were up-regulated or down-regulated. The supplementation experiment with BCAAs confirmed that 0.2 g/L of L-Ile and 0.5 g/L of L-Val increased natamycin yield by 17.6% and 37.8%, respectively. Fungal elicitor also up-regulated the transcriptional levels of most of the enzymes associated with the biosynthesis of natamycin and two important transcription regulators ( pimR and pimM ). To confirm the accuracy of RNA-Seq, the results of qPCR showed that these gene expression levels were in agreement with the transcription changes by RNA-Seq. Conclusion In this study, the change of transcriptional levels in S. natalensis HW-2 under treated with the fungal elicitor was firstly reported. The major finding of our comparative transcriptome analysis is that the fungal elicitor improves the supply of precursor, and alters the expression of natamycin related genes and regulator of secondary metabolism. From our results, we conclude that regulatory alterations are important factors for the enhancement of natamycin.

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Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Cited by 25 publications

References 40 publications

Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production

Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production

Enhanced Bacitracin Production by Systematically Engineering S-Adenosylmethionine Supply Modules in Bacillus licheniformis

Transcriptome analysis of the regulation of natamycin biosynthesis in Streptomyces natalensis HW-2 by fungal elicitor

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