​Nat Commun Bian Xiaoying's team develops a new type of gram-negative bacterial chassis to promote the mining of natural products

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The genome contains a large number of hidden or "silent" natural product biosynthetic gene clusters, and their coded products represent a large number of unexplored active natural product resources of biologically active metabolites [1]
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The heterologous expression of natural product biosynthetic gene clusters in high-efficiency chassis cells is beneficial to increase yield and discover new natural products [2]
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Chassis construction has always been the core of synthetic biology and green biological manufacturing of natural products
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However, due to the lack of efficient bottom plate bacteria, the mining of recessive biosynthetic gene clusters in gram-negative bacteria has been limited for a long time
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In previous studies, Burkholderia DSM 7029 (now identified as Schlegelella brevitalea) of the β-Proteobacteria grew faster, had easy genetic manipulation, and was rich in secondary metabolism to successfully express multiple myxobacteria-derived non-ribosomal polypeptides/ Polyketone complex natural products, such as the anti-cancer drug epothilone, have the potential to become a universal natural product chassis [3-5]
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However, the bacterium undergoes severe autolysis in the early stage of liquid culture, which significantly affects the biomass and limits the heterologous expression yield of the target compound, which hinders its further application
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Although adding a large amount of sucrose to the medium can delay cell death and prolong the fermentation cycle to a certain extent [6], the autolysis mechanism of this bacterial cell is still unclear, and whether genome modification can be used to delay autolysis remains to be verified
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On July 23, 2021, Bian Xiaoying's research group at Shandong University published a research paper entitled Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria in Nature Communications.
This research uses the efficient genome editing constructed in the previous period.
The technology simplifies the rational genome of Burkholderia DSM 7029.
The strains with deleted transposons and prophage showed optimized growth phenotypes, significantly reduced early cell autolysis, and initially revealed the mechanism of autolysis
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Using this optimized chassis, the production of six heterologous natural products such as the anticancer drug epothilone has been significantly increased, and a new type of non-ribosomal peptide compound in Chitinomonas has been identified, which is superior to commonly used leathers.
Blue-negative bacterial chassis-Escherichia coli and Pseudomonas putida
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Therefore, a gram-negative bacteria chassis with independent intellectual property rights has been constructed, which is expected to become a high-quality chassis for expressing gram-negative bacteria gene clusters for natural product mining and transformation
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In this study, the author rationally designed two parallel deletion routes through bioinformatics analysis, and used the genome editing technology established in the early stage of the research group to continuously delete large fragments of genome sequences [5], constructing a simplification of multiple gene combinations.
The mutant strain (Figure 1)
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Figure 1.
Flow chart for the construction of simplified genome mutants
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The left genome map represents the wild-type Burkholderia DSM 7029 genome.
The biosynthetic gene cluster region to be deleted is indicated by a black bold short line, and the target deletion transposase, genome island, prophage and other regions are represented by two black counterparts.
Triangular representation
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In the first genomic simplification route, a series of DC mutant strains were obtained after successive deletion of multiple endogenous secondary metabolic biosynthetic gene clusters.
Although the metabolic background was significantly reduced, the cell growth of DC5 ~ DC7 mutant strains was obviously affected.
It affects and shows cell fragility in the early stage of liquid culture (Figure 2)
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On the contrary, in the second simplified route, after deleting multiple transposases, genome islands, prephages and adjacent non-essential genes, DT series mutant strains were obtained, and DT series mutant strains showed obviously optimized growth characteristics.
And significantly reduced cell autolysis (Figure 3), and has a smaller cell morphology and higher electrotransformation efficiency of foreign DNA
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Therefore, on the basis of DT7, continue to delete the secondary metabolism gene cluster to construct DT8~DT10 with reduced metabolic background
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Figure 2.
Physiological and phenotypic identification of the DC series of genome-simplified mutants
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a.
Growth curve of wild-type Burkholderia DSM 7029 and mutant strains DC1~DC7; b.
Wild-type Burkholderia DSM 7029 and mutant strains DC4~DC7 were cultured in CYMG liquid medium for 24 hours, 48 ​​hours and 72 hours Cell morphology under a scanning electron microscope at hour
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The author then studied the optimized growth phenotype and reduced autolysis in the DT series of mutant strains
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Since the quorum sensing system is widespread in the genus Burkholderia, studies have shown that it plays an important role in key cell life processes such as nutrient acquisition and cell homeostasis maintenance.
Then the author deleted the genome of the DT series of mutant strains.
Bioinformatics analysis of the region, single knockout of 16 putative cell quorum sensing-related and autolysis-related regulatory factors, preliminary identification of negative regulatory factors related to cell growth in DSM 7029, such as Tet/AcrA family transcription regulatory factors and LysR family transcriptional regulators
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At the same time, based on single knockout experiments, the authors proposed that the significant reduction of autolysis in DT6~DT10 mutant strains may be related to the deletion of lyase-encoding genes or autolysis-related negative regulatory factors, such as the identified lyase and XRE family transcription Regulatory factor
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In addition, the authors compared the relative yields of six natural products derived from Burkholderia or myxobacteria, such as the anticancer drug epothilone, by the method of site-specific recombination-mediated integration of foreign gene clusters.
And based on the fact that the output of heterologous expression in the DT series of mutant strains is higher than the output of heterologous expression in DSM 7029 wild-type and the other two widely used Gram-negative chassis bacteria Escherichia coli and Pseudomonas putida, it is demonstrated that DT6 ~DT10 gene combination simplifies the excellent chassis bacteria characteristics of mutant strains.
Among them, DT8~DT10 mutant strains have lower metabolic background and more efficient heterologous expression ability, and can be used as preferred chassis bacteria for application
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Finally, a cryptic biosynthetic gene cluster chm derived from the gram-negative bacterium Chitinimonas koreensis DSM 17726 was successfully expressed heterologously in the DT series of mutant strains, and the yield of heterologous expression was higher than that of the wild-type DSM 7029 Compared with the original strain DSM 17726, it is significantly improved, and a series of new natural products, chitinimides AH, have been identified
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This study extends the detoxin/rimosamide biosynthetic gene cluster family to Gram-negative Proteobacteria, and the anti-antibiotic activity shown by these natural products indicates that these microorganisms have complex ecological significance.
The biological functions and activities of chitinimides remain Need further research
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In this study, a series of genetic combinations were constructed to simplify Burkholderia DSM 7029.
The chassis showed significantly improved growth characteristics and optimized the efficiency of heterologous expression of natural products of gram-negative bacteria, expanded the chassis of gram-negative bacteria, and It has the potential to become a universal natural product chassis, which further promotes the targeted mining of new natural products based on heterologous expression of gene clusters and the increase in the yield of target products
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Moreover, this study once again proves that the combined deletion of non-essential genes such as transposons, inserted sequences, and prophage is a feasible method to construct the optimal chassis [7], which provides an example for the construction of more bacterial chassis and cell factories
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Original link: https:// Platemaker: 11 References 1 Rutledge, PJ & Challis, GL Discovery of microbial natural products by activation of silent biosynthetic gene clusters.
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Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029.
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Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.
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Discovery of recombinases enables genome mining of cryptic biosynthetic gene clusters in Burkholderiales species.
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Reassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.
ACS Synth Biol 9, 2009-2022, doi:10.
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7 Martinez-Garcia, E.
& de Lorenzo, V.
Pseudomonas putida in the quest of programmable chemistry.
Curr Opin Biotechnol 59, 111-121, doi:10.
1016/j.
copbio.
2019.
03.
012 (2019).
Reprinting instructions [Non-original article] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting without permission is prohibited , The author has all legal rights, offenders must be investigatedReassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.
ACS Synth Biol 9, 2009-2022, doi:10.
1021/acssynbio.
0c00100 (2020).
7 Martinez-Garcia, E.
& de Lorenzo, V.
Pseudomonas putida in the quest of programmable chemistry.
Curr Opin Biotechnol 59, 111-121, doi:10.
1016/j.
copbio.
2019.
03.
012 (2019).
Reprint Instructions [Non-original article] The copyright of this article belongs to the author of the article, and personal sharing is welcome.
Reprinting is prohibited without permission, the author has all legal rights, offenders must be investigatedReassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.
ACS Synth Biol 9, 2009-2022, doi:10.
1021/acssynbio.
0c00100 (2020).
7 Martinez-Garcia, E.
& de Lorenzo, V.
Pseudomonas putida in the quest of programmable chemistry.
Curr Opin Biotechnol 59, 111-121, doi:10.
1016/j.
copbio.
2019.
03.
012 (2019).
Reprint Instructions [Non-original article] The copyright of this article belongs to the author of the article, and personal sharing is welcome.
Reprinting is prohibited without permission, the author has all legal rights, offenders must be investigated
.