Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production

Song, Kejing; Wei, Liang; Liu, Jiao; Wang, Junhua; Qi, Haishan; Wen, Jianping

doi:10.1007/s00253-017-8242-4

Cited by 28 publications

(37 citation statements)

References 40 publications

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“…Other examples include LysR family regulator for ascomycin production in S. hygroscopicus var. ascomyceticus (Song et al, 2017), PAS-LuxR family regulator for wuyiencin production in Streptomyces wuyiensis CK-15 , and Crp/Fnr family regulator for leinamycin production in Streptomyces atroolivaceus (Huang et al, 2016).…”

Section: Enhancing Antibiotic Production By Overexpression Of Positivmentioning

confidence: 99%

The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining

Xia

et al. 2020

Front. Microbiol.

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Streptomyces is taken as an important resource for producing the most abundant antibiotics and other bio-active natural products, which have been widely used in pharmaceutical and agricultural areas. Usually they are biosynthesized through secondary metabolic pathways encoded by cluster situated genes. And these gene clusters are stringently regulated by interweaved transcriptional regulatory cascades. In the past decades, great advances have been made to elucidate the regulatory mechanisms involved in antibiotic production in Streptomyces. In this review, we summarized the recent advances on the regulatory cascades of antibiotic production in Streptomyces from the following four levels: the signals triggering the biosynthesis, the global regulators, the pathway-specific regulators and the feedback regulation. The production of antibiotic can be largely enhanced by rewiring the regulatory networks, such as overexpression of positive regulators, inactivation of repressors, fine-tuning of the feedback and ribosomal engineering in Streptomyces. The enormous amount of genomic sequencing data implies that the Streptomyces has potential to produce much more antibiotics for the great diversities and wide distributions of biosynthetic gene clusters in Streptomyces genomes. Most of these gene clusters are defined cryptic for unknown or undetectable natural products. In the synthetic biology era, activation of the cryptic gene clusters has been successfully achieved by manipulation of the regulatory genes. Chemical elicitors, rewiring regulatory gene and ribosomal engineering have been employed to crack the potential of cryptic gene clusters. These have been proposed as the most promising strategy to discover new antibiotics. For the complex of regulatory network in Streptomyces, we proposed that the discovery of new antibiotics and the optimization of industrial strains would be greatly promoted by further understanding the regulatory mechanism of antibiotic production.

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Section: Enhancing Antibiotic Production By Overexpression Of Positivmentioning

confidence: 99%

The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining

Xia

et al. 2020

Front. Microbiol.

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“…With the exception of fkbG , most of the differentially expressed genes had increased expression levels in DMSO_H compared to levels in DMSO_N. Notably, the gene transcriptional directions for fkbG and fkbH/I/J/K are different in the ascomycin gene cluster (Song et al, ), and fkbG may be located at a different transcriptional position compared with the other genes. Therefore, fkbG expression might be regulated by other factors.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, this gene might have the same function in ascomycin production. The LysR family transcriptional regulator encoded by fkbR1 was reported to be a positive regulator of ascomycin overproduction, which is negatively autoregulated and affects ascomycin biosynthesis mainly in the exponential phase of cultivation (Song et al, ). The relative upregulation of fkbR1 in the DMSO_H versus DMSO_N and DMSO_L versus DMSO_N comparisons may indicate increased activation with increasing DMSO concentration (Table ).…”

Section: Resultsmentioning

confidence: 99%

“…However, the biosynthesis of secondary metabolites is regulated not only by the precursor synthetic pathways but also by the complex intracellular regulatory networks (e.g., pathway‐specific regulators and global/pleiotropic regulators). For instance, by engineering the LysR family transcriptional regulators fkbR1 and fkbE , the final ascomycin yield increased by 69.9% (Song et al, ). Nevertheless, the achievable yield of ascomycin remains low, greatly limiting large‐scale industrial production.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway‐engineering strategies

Wang

Yuan

et al. 2019

Biotech & Bioengineering

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Ascomycin (FK520) is a macrocyclic antibiotic that also exhibits antifungal and immunosuppressive activity. However, its relatively low titer and yield have hampered commercial application. Here, we have successfully constructed an efficient ascomycinproducing strain of Streptomyces hygroscopicus with high titer and yield, using a novel combinatorial engineering approach based on the identification of targets involved in both metabolic and transcriptional regulation. First, we investigated the effects of different chemicals on ascomycin accumulation and found that dimethyl sulfoxide best stimulated ascomycin overproduction. We next compared intracellular metabolic and transcriptional profiles after dimethyl sulfoxide and control treatments and identified potential target genes (zwf and aroA, involved in metabolic precursor pathways; and luxR, iclR, fadR, and fkbN, involved in transcriptional regulation). These candidate genes were then engineered to produce strains with individual and combinatorial overexpression. Combined overexpression of aroA, fkbN, and luxR resulted in the highest yield of ascomycin (1258.30 ± 33.49 mg/L), 4.12-fold higher than the control yield (305.60 ± 16.90 mg/L). This integrative multilevel approach identified novel determinants involved in both metabolic and transcriptional regulation, resulting in the diversion of carbon flux towards ascomycin accumulation. This approach could be applied to boost the production of a variety of useful bacterial metabolites.

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“…The combined genetic manipulation of target genes fkbO and pyc predicted by 13 C-metabolic ux analysis increased the FK520 production by 24.2% [10]. And the combined overexpression of the pathway-speci c regulatory gene fkbR1 and its target gene fkbE increased the FK520 production by 69.9% [11]. However, the carbon sources supply for the synthesis of FK520 precursors in the late phase of fermentation is still an urgent matter to be resolved.…”

Section: Introductionmentioning

confidence: 99%

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

Wang

Yin

Wang

et al. 2020

Preprint

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Background: Ascomycin is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. As a secondary metabolite, the production of ascomycin is often limited by the shortage of precursors during the late fermentation phase. Polyhydroxybutyrate is an intracellular polymer accumulated by various microorganisms. The development of polyhydroxybutyrate as a useful carbon reservoir for precursor synthesis is of great significance for improving the yield of ascomycin.Results: The fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate were respectively correlated with the growth of strain and the production of ascomycin. The co-overexpression of exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both strain biomass and ascomycin yield. Comparative transcription analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, and the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing metabolic flux of primary pathways. The comparative analysis of cofactor concentration reflected that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH pool. Under the condition of low sugar content in the later exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of co-overexpression strain was increased to 626.30 mg/L, 2.11-fold higher than that of parent strain in the initial medium (296.29 mg/L). Conclusions: This paper reported for the first time that the polyhydroxybutyrate was beneficial to the growth of strain and the production of ascomycin by working as an intracellular carbon reservoir, storing as polymers when carbon sources are abundant and depolymerizing to monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provided a new strategy for the high yield of other secondary metabolites.

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Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production

Cited by 28 publications

References 40 publications

The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining

The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining

Generation of Streptomyces hygroscopicus cell factories with enhanced ascomycin production by combined elicitation and pathway‐engineering strategies

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

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