Amphotericin B biosynthesis in Streptomyces nodosus: quantitative analysis of metabolism via LC-MS/MS based metabolomics for rational design

Abstract

Background: Amphotericin B (AmB) is widely used against fungal infection and produced mainly by Streptomyces nodosus. Various intracellular metabolites of S. nodosus were identified during AmB fermentation, and the key compounds that related to the cell growth and biosynthesis of AmB were analyzed by principal component analysis (PCA) and partial least squares (PLS).

Results: Rational design that based on the results of metabolomics was employed to improve the AmB productivity of Streptomyces nodosus, including the overexpression of genes involved in oxygen-taking, precursor-acquiring and product-exporting. The AmB yield of modified strain S. nodosus VMR4A was 6.58 g/L, which was increased significantly in comparison with that of strain S. nodosus ZJB2016050 (5.16 g/L). This was the highest yield of AmB reported so far, and meanwhile, the amount of by-product amphotericin A (AmA) was decreased by 45%. Moreover, the fermentation time of strain S. nodosus VMR4A was shortened by 24 h compared with that of strain. The results indicated that strain S. nodosus VMR4A was an excellent candidate for the industrial production of AmB because of its high production yield, low by-product content and the fast cell growth.

Conclusions: This study would lay the foundation for improving the AmB productivity through metabolomics analysis and overexpression of key enzymes.

Keywords: Amphotericin B; Metabolomics; Overexpression; Streptomyces nodosus.

Fig. 1

Structures and the antibiotic biosynthesis gene cluster of amphotericin. a Structures of amphotericin B and amphotericin A, which are different in the reduction of C28–C29 double bond. b The amphotericin biosynthesis gene cluster is organized with PKS genes, post-PKS modification genes, transporter and regulator genes and other ORF genes, which are described by white arrow, white arrow with line, grey arrow and black arrow, respectively

Fig. 2

Fermentation profiles for strain S. nodosus ZJB2016050. Four profiles are illustrated in the line chart, including the yield of AmB, dry cell weight, pH and residual glucose. The whole process could be divided into four phases, lag phase (0–24 h), exponential phase (24–108 h), stationary phase (108–132 h) and decline phase (132–168 h). Each value is a mean of three experiments. Error bars show standard derivation among three experiments

Fig. 3

PCA and PLS-DA analysis of intracellular metabolites at different fermentation time points. The samples were withdrawn from the cultivation at 24, 72, 120 and 156 h. a PCA scores scatter plot in positive ion scan modes. b PCA scores scatter plot negative ion scan modes. c PLS-DA scores scatter plot in positive ion scan modes. d PLS-DA scores scatter plot in negative ion scan modes. In order to assess the accuracy and stability of the equipment state during detection and collection process, the quality control samples (the mixture of all samples) were prepared in advance, and then were carried out every 10 samples

Fig. 4

The relative abundances of various intracellular metabolites in different fermentation period. Metabolites were analyzed to investigate the differences during the whole fermentation process of S. nodosus, including metabolism of amino acid, sugar, fatty acid, terpenoid backbone, folate biosynthesis and the other secondary metabolites. a Amino acid metabolism, b sugar metabolism and central metabolic pathway, c fatty acid biosynthesis, d terpenoid backbone biosynthesis, e folate biosynthesis and one carbon pool by folate, f secondary metabolites and antibiotics. Metabolites annotation have been checked by authentic standards (Glucose 6-phosphate, Glycerol, serine, cysteine, SAM, Farnesol, THF-polyglutamate, Amphotericin), Red * indicates authentic standards. The error bars represent standard deviations of five values

Fig. 5

AmB production associated with genes overexpression and the fermentation time courses. a AmB production associated with genes overexpression in various engineered strains, the genetically engineered strains wereall constructed from primitive stain, S. nodosus ZJB2016050. ZJB2016050 represents strain S. nodosus ZJB2016050, pJTU1278 represents strain S. nodosus ZJB2016050 with plasmid pJTU1278. vhb, metK, amphRI, amphRIV, amphGH, amphG and araC represent overexpression of gene vhb, metK, amphRI, amphRIV, amphH, amphG and araC, respectively, in strain S. nodosus ZJB2016050 with plasmid pJTU1278. VMR4A and VMR4HGA were strain overexpressed four genes (vhb, metK, amphRIV and araC connected by ermE*p) and six genes (vhb, metK, amphRIV, amphH, amphG and araC connected by ermE*p), respectively. Samples were collected from soluble fermentation at 144 h, and the AmB concentration and ration of AmA were detected and analyzed, respectively. b Fermentation time course for strain S. nodosus ZJB2016050, pJTU1278, VMR4A and VMR4HGA. The ZJB2016050 and pJTU1278 were primitive strain and the strain with empty vector, respectively. VMR4A and VMR4HGA were strain overexpressed four genes (vhb, metK, amphRIV and araC connected by ermE*p) and six genes (vhb, metK, amphRIV, amphH, amphG and araC connected by ermE*p), respectively. Each value is a mean of three experiments. Error bars show standard derivation among three experiments. Symbol ‘*’ means the experimental strain compared with the original strain ZJB2016050 and ^× means the experimental strain compare with strain with vector pJTU1278 (*p < 0.05, **p < 0.01, ^×p < 0.05 and ^××p < 0.01)