Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the "pathway redundancy" in metabolic networks

Abstract

Background: Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the "pathway redundancy" of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer.

Results: Here we report the genetic characterization of CPOAA biosynthesis with revealing a "pathway redundancy" in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles.

Conclusions: It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the "pathway redundancy", more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the "pathway redundancy" in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies.

Figure 1

The proposed CPOAA biosynthetic pathway (A) and Genetic organization of the polyoxin biosynthetic gene cluster (B). The initial two steps involved in CPOAA biosynthesis are overlapped with those of L-Arginine pathway.

Figure 2

Heterologous expression of the pJTU4620 derivatives (with individual mutation of polN, polM and polL) in S. lividans TK24. (A) Schematic representation for the construction of pJTU4620 derivatives, the genes polL-N were independently in-frame-deleted in pJTU4620. (B) Bioassay for the related S. lividans TK24 recombinants, the sample of pJTU4620 (TK24 containing pJTU4620), pJTU4620/ΔpolN, pJTU4620/ΔpolM and pJTU4620/ΔpolL were correspondingly spotted as 1-4; TK24 containing pSET152 (spot 5) was used as negative control. (C) HPLC analysis of the metabolites individually produced by the S. lividans TK24 recombinants. The authentic polyoxin standards (ST) and the sample of TK24 containing pJTU620 (4620) were used as positive controls, and TK24 containing pSET252 was used as negative control. The samples of TK24 bearing 4620 derivatives were individually indicated as 4620/ΔpolN, pJTU4620/ΔpolM and 4620/ΔpolL. POL: polyoxin.

Figure 3

Targeted inactivation of argA in E. coli and mutant complementation by polN. (A) Representational map for construction of the CH3 mutant. (B) Identification of CH2 mutants. As 1.1-kb argA fragment was replaced by 1.0-kb aac(3)IV gene, the PCR product for CH3 mutants was ca. 1.5-kb, while the wild type produces 1.6-kb PCR product. (C) Complementation of CH3 mutant by polN. CH3 mutant of E. coli BL21(DE3) (1) and CH3 mutant containing pET28a (2) were used as negative controls, while CH3 mutant containing pJTU2838 with inserted polN was indicated as (3). The final concentration for arginine and thymidine used in this experiment is 50 μg/ml and 200 μg/ml, respectively, and the liquid cultures were incubated at 37°C for 90 h.

Figure 4

Targeted inactivation of polP. (A) Schematic representation for the construction of CY7 mutant. (B) PCR confirmation of the CY7 mutants, as a 1.1-kb aac(3)IV fragment replaced 495-bp of polP, the CY7 mutants give 1.6-kb PCR product, while WT of S. cacaoi produces 1.0-kb product. (C) Bioassay of the metabolites produced by CY7 mutant, the sample of S. cacaoi wild type indicated as (1) was used as positive control, and the samples from CY7 mutants were indicated as (2-4). 35 μl sample (supernatant) was used for all bioassays in this study. (D) HPLC analysis of the metabolites produced by CY7 mutant. Polyoxins authentic standard (ST) and sample from wild-type of S. cacaoi (WT) were used as positive control, and the sample from CY7 mutant was indicated as CY7.

Figure 5

In frame deletion of argB in S. coelicolor A3(2) and mutant complementation by polP. A. Schematic representation for construction of CX2 mutant; B. PCR identification of CX2 mutant. As ca. 0.4-kb of argB was in frame deleted, the wild type of S. coelicolor A3(2) gives a 1.1-kb product, and the products of CX2 mutant are 0.68-kb in size. C. Plate grown experiments for CX2 mutant and its complemented strains, S. coelicolor A3(2) wild type (1) and CX2 mutant complemented by argB (S. cacaoi) in pJTU4713 (5) were used as positive controls, and CX2 mutant containing empty vector, pJTU2170, was used as negative control (3); CX2 mutant (2) and its complemented strain by polP in pJTU2870 (4) were indicated as parallels.

Figure 6

Natural argB plays essential roles for both polyoxin production and L-arginine biosynthesis. (A) PCR identification of CY22 mutant, the construction process is identical to that of CY7 mutant except that the CY21 mutant was used as start strain, and PCR product of CY22 mutant (1) was compared with that of CY21 mutant (2). (B) Growth phenotype of CY22 and its related complemented strains in minimal medium. CY7 mutant was used as positive control (1), and CY22 mutant (2) as well as its complemented strain (CY22/pJTU2170) with an empty vector (3) were selected as negative controls, CY22/pJTU4713 (argB) (4) and CY22/pJTU2870 (polP) (5) were detected as parallels. (C) Bioassay for the metabolites produced by CY22 and its related complemented strains. Samples of CY21 mutant (1), CY22 mutant (2), and CY22/pJTU2170 (3) were detected in parallel with those of CY22/pJTU4713 (argB) (4) and CY22/pJTU2870 (polP) (5). (D) HPLC analysis of the metabolites produced by the CY22 mutant and its related strains. Polyoxin authentic standard (ST) and metabolites produced by S. cacaoi wild type (WT) were used as positive controls, while the metabolites produced by CY21, CY22, CY22/pJTU2170 were utilized as negative controls; the samples of CY22/argB (pJTU4713) and CY22/polP (pJTU2870) were detected as parallels.

Figure 7

Time-course bioassay and transcriptional analysis of S. cacaoi CY21 strain (argB mutant). (A) Time-course bioassay for CY21 and WT strains of S. cacaoi; (B) Time-course transcriptional analysis of S.cacaoi CY21 strain, and RT-PCR was used for time-course transcriptional analysis of CY21 strain. “W” indicates wild type of S. cacaoi, and “M” means CY21 mutant of S. cacaoi. The methods for the time-course bioassay and time-course transcriptional analysis of S. cacaoi CY21 strain were described in Additional file 1.