Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis

Wu, Zheng; Gao, Wenli; Zhou, Shaotong; Zhao-lin, Wen; Ni, Xianpu; Xia, Huanzhang

doi:10.1016/j.micres.2017.06.006

Cited by 7 publications

(3 citation statements)

References 18 publications

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“…Our previous study also identified dozens of constitutive promoters that exhibited stronger activity than ermE* in Streptomyces (Liu et al., 2016). However, in previous work, there was no report on the determination of promoter strength in Micromonospora , and researchers could only roughly use hrdB and ermE* for gene overexpression (Ni et al., 2016; Wu et al., 2017). In this study, we tested these promoters’ strengths in Micromonospora by measuring the relative activity of the reporter gene gusA which was placed downstream of each candidate promoter to provide more selectable promoter elements for subsequent gene overexpression.…”

Section: Resultsmentioning

confidence: 99%

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS

Chang

Chai

Ding

et al. 2019

Metabolic Engineering Communications

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In pharmaceutical industry, isepamicin is mainly manufactured from gentamicin B, which is produced by Micromonospora echinospora as a minor component of the gentamicin complex. Improvement of gentamicin B production through metabolic engineering is therefore important to satisfy the increasing demand for isepamicin. We hypothesized that gentamicin B was generated from gentamicin JI-20A via deamination of the C2’ amino group. Using kanJ and kanK as the gene probes, we identified the putative deamination-related genes, genR and genS, through genome mining of the gentamicin B producing strain M. echinospora CCTCC M 2018898. Interestingly, genR and genS constitute a gene cassette located approximately 28.7 kb away from the gentamicin gene cluster. Gene knockout of genR and genS almost abolished the production of gentamicin B in the mutant strain, suggesting that these two genes, which are responsible for the last steps in gentamicin B biosynthesis, constitute the missing part of the known gentamicin biosynthetic pathway. Based on these finding, we successfully constructed a gentamicin B high-yielding strain (798 mg/L), in which an overexpression cassette of genR and genS was introduced. Our work fills the missing piece to solve the puzzle of gentamicin B biosynthesis and may inspire future metabolic engineering efforts to generate gentamycin B high-yielding strains that could eventually satisfy the need for industrial manufacturing of isepamicin.

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Section: Resultsmentioning

confidence: 99%

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS

Chang

Chai

Ding

et al. 2019

Metabolic Engineering Communications

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“…46). The substantial titer improvement of 1 with highly efficient heterologous G-1-PTs [31][32][33] further demonstrated that overexpression of enzymes that divert primary metabolites into secondary metabolism is a useful strategy 41 (Fig. 6e).…”

Section: Discussionmentioning

confidence: 92%

A severe leakage of intermediates to shunt products in acarbose biosynthesis

et al. 2020

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The α-glucosidase inhibitor acarbose, produced by Actinoplanes sp. SE50/110, is a well-known drug for the treatment of type 2 diabetes mellitus. However, the largely unexplored biosynthetic mechanism of this compound has impeded further titer improvement. Herein, we uncover that 1-epi-valienol and valienol, accumulated in the fermentation broth at a strikingly high molar ratio to acarbose, are shunt products that are not directly involved in acarbose biosynthesis. Additionally, we find that inefficient biosynthesis of the amino-deoxyhexose moiety plays a role in the formation of these shunt products. Therefore, strategies to minimize the flux to the shunt products and to maximize the supply of the amino-deoxyhexose moiety are implemented, which increase the acarbose titer by 1.2-fold to 7.4 g L −1 . This work provides insights into the biosynthesis of the C 7 -cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.

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“…The industrial production of etimicin requires a significant quantity of high-purity gentamicin C1a. As a result, many researchers have employed genetic engineering techniques to develop strains that produce high yields of single-component C1a [6,[19][20][21].…”

Section: Determination Of Sugar Amino Nitrogen Germ Concentration And...mentioning

confidence: 99%

Proteomic Analysis of the Effect of CaCl2 and Sodium Citrate on Gentamicin Biosynthesis of Micromonospora echinospora SIPI-GM.01

Yang,

Lin,

et al. 2023

Fermentation

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The clinical antibiotic gentamicin is a mixture of several difficult-to-separate components, the minor group of which is gentamicin C1a, a precursor for the synthesis of the high-efficacy and low-toxicity antibiotic etimicin. This study aimed to achieve the high production of gentamicin as well as gentamicin C1a. In this study, the influence of organic and inorganic salts on the gentamicin production was screened and label-free proteomics was used to determine the mechanisms responsible for the effects. In 25 L fermentation experiments, the addition of 0.1% CaCl2 and 0.3% sodium citrate increased gentamicin titers by 11.5% (2398 μg/mL vs. 2150 μg/mL), while the C1a ratio increased from 38% to 42%. The results showed that CaCl2 downregulated the synthesis and metabolism of the tetrapyrrole pathway and the GenK protein (0.08-fold) in the gentamicin synthesis pathway, whereas sodium citrate downregulated key proteins in the glycosylation pathway and tricarboxylic acid pathway. Thus, CaCl2 caused changes in methylation during the synthesis of gentamicin, increasing the proportion of gentamicin C1a. In contrast, sodium citrate inhibited primary metabolism to promote the production of secondary metabolites of gentamicin. This study provided a basis for the co-production of gentamicin C1a mono-component and gentamicin multicomponent.

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Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis

Cited by 7 publications

References 18 publications

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS

A severe leakage of intermediates to shunt products in acarbose biosynthesis

Proteomic Analysis of the Effect of CaCl2 and Sodium Citrate on Gentamicin Biosynthesis of Micromonospora echinospora SIPI-GM.01

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