Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the “pathway redundancy” in metabolic networks
© Chen et al.; licensee BioMed Central Ltd. 2013
Received: 24 September 2013
Accepted: 24 November 2013
Published: 7 December 2013
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.
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.
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.
The structure of polyoxin is composed of three building blocks including a nucleoside skeleton, polyoximic acid (POIA) and carbamoylpolyoxamic acid. Previous studies with feeding experiments have demonstrated that the three building blocks were individually originated from uridine (or UMP), L-isoleucine and L-glutamate . Previously, the polyoxin biosynthetic gene cluster was cloned and well characterized, and the entire polyoxin gene cluster was revealed to consist of 20 genes (Figure 1B), of them, three genes (polF, polC and polE) were proposed to be involved in POIA biosynthesis, and five genes (polL-P) were assigned as the roles for CPOAA biosynthesis [9, 10].
As proposed in previous studies, CPOAA is formed by first transfer of an N-acetyl group to L-glutamate by PolN to form N-acetyl-L-glutamate, followed by phosphorylation to form N-acetyl-L-glutamate phosphate by PolP before stepwise reduction, deacylation, transcarbamoylation and hydroxylation (Figure 1A) . Remarkably, the role of PolO as carbamoyltransferase catalyzing α-amino-δ-hydroxyvaleric acid (AHV) to generate α-amino-δ-hydroxyvaleric carbamoylhydroxyvaleric acid (ACV) was unambiguously demonstrated by in vivo and in vitro experiments . According to the proposed pathway, the first two steps were identical to those for the L-arginine biosynthetic pathway, in which the initial steps involve catalyzing L-glutamate to form N-acetyl-L-glutamyl-5-phosphate by sequential acylation and phosphoralation [9, 11]. As an important amino acid, L-arginine plays key roles in many metabolic pathways, either primary or secondary . The L-arginine biosynthetic pathway was distinctly characterized in bacteria including E. coli and Streptomyces . In E. coli, there is a linear biosynthetic pathway for L-arginine biosynthesis , however, many other bacteria such as Streptomyces harbor cyclic L-arginine biosynthetic pathway [11–13], in which the N-acyl group of the intermediate N-acetyl-L-ornithine could be recycled as substrate for ArgA, therefore, the protein, ArgJ, in Streptomyces displays bifunctional roles for the acylation and deacylation .
Here we describe the genetic characterization of CPOAA biosynthesis with unveiling of the “pathway redundancy” in metabolic networks. In silico and genetic analysis of the polyoxin gene cluster demonstrated that five genes (polL-P) were involved in CPOAA biosynthesis; moreover, the inter-connections between metabolic pathways of polyoxin and L-arginine were systematically characterized by in vivo genetic experiments. All these will set the stage for the understanding of the “pathway redundancy” in metabolic networks, and pave the way for rational designing and optimizing the polyoxin biosynthetic pathway so as to increase antibiotic production via the strategies of pathway engineering.
In silico analysis of the candidate genes for CPOAA biosynthesis
In silico analysis shows that the five genes (polL-P) in the pol cluster seem to be involved in biosynthesis of CPOAA. Among them, polL encodes a protein exhibiting no detectable homology to any proteins in the database, suggesting its obscure and unique catalytic mechanism for CPOAA biosynthesis; PolM is a 255-aa protein with considerable homology to short-chain dehydrogenase of Pseudomonas aeruginosa PAO1. Short-chain dehydrogenase is a very large family of enzymes, most of which are known to be NAD- or NADP-dependent oxidoreductases. The protein PolN shows low homology (32% identity) to amino-acid N-acetyltransferase of Neisseria mucosa ATCC 25996. PolP exhibits 54% identity to the acetylglutamate kinase of Frankia alni ACN14a, implying the similar catalytic mechanism of PolP in CPOAA biosynthesis. PolO displays significant homology to NodU, a carbamoyltransferase of Sinorhizobium sp , whose function was unambiguously confirmed as a carbamoyl transferase by previous in vitro experiments .
Independent mutation of polL-N led to the accumulation of thymine polyoxin I
polN possesses the ability to restore the growth phenotype for E. coli argA mutant
Targeted disruption of polP partially affects polyoxin production
To further confirm the identities of the two intermediates (N1 and N2), both peaks were analyzed by LC/MS, and the results showed that the peaks at 33.1 min and 36.5 min could produce the [M+H]+ ions at m/z = 427.3 and 411.4, respectively, which corresponds to the authentic standards of polyoxin I and thymine polyoxin I (Additional file 1: Figure S4), moreover, MS/MS fragmentation pattern of the two peaks were in consistent with the standards (Additional file 1: Figure S4), suggesting that polP is not essential for CPOAA biosynthesis but only for its maximal production, likewise, indicating a homolog existing to rescue the polP mutation.
polP is capable of complementing argB mutant of Streptomyces coelicolor
Natural argB plays essential roles for both polyoxin production and L-arginine biosynthesis in S. cacaoi
To investigate whether argB mutant of S. cacaoi could survive the growth environment without exogenous L-arginine, the mutant (CY21) was constructed (Additional file 1: Figure S6). In contrast with our expectations, the CY21 mutants were unable to grow without added L-arginine (Additional file 1: Figure S6). Furthermore, the growth phenotype was entirely complemented by an introduced argB (CY21/pJTU4713) (Additional file 1: Figure S6), suggesting that the natural polP is not capable of complementing the CY21 mutant, simultaneously excluding the possibility of a polar effect (or frameshift mutation).
To further see if the phenotype of CY7 was conferred by argB, the CY22 mutant and its complemented strains were inoculated for fermentations, after that, the preprocessed broth were subjected to bioassay analysis, and the results showed that the samples of CY22 and CY22/pJU2170 have abolished bioactivity against the indicator strain, Trichosporon cutaneum (Figure 6C). More interestingly and unexpectedly, the sample of the CY21 mutant has also been deprived of the bioactivity against the indicator strain, however, the CY22 mutant correspondingly complemented by polP and argB regained the bioactivity (Figure 6C). Further HPLC analysis indicated that the samples of CY21, CY22 and CY22/pJTU2170 were not capable of producing the distinctive peaks of either polyoxin A or polyoxin H as indicated by the standards at 34.8 min and 40.9 min, respectively, while the polP or argB complemented strains restored the abilities to produce the characteristic peaks at corresponding positions (Figure 6D).
Nucleoside antibiotics are a family of secondary metabolites whose biosynthetic precursors are originated from primary metabolisms including nucleotide (nucleoside), amino acids and saccharides [1, 2, 17]. Previous experiments assigned the biosynthetic precursors for the three building blocks (nucleoside skeleton, POIA and CPOAA) of polyoxin as UMP, L-isoleucine and L-glutamate, respectively [2, 9, 18–20]. As for CPOAA biosynthesis, the former labeling results led to a proposal that the biosynthesis was initiated by catalyzing L-glutamate to produce L-glutamate-γ-semialdehyde, which was finally converted to CPOAA with stepwise reactions [19, 21].
The polyoxin biosynthetic gene cluster was previously cloned and sequenced , and further bioinformatic insights into the polyoxin biosynthetic gene cluster contradict to the previously-deduced pathway. For one thing, the hypothetic L-glutamate-γ-semialdehyde in CPOAA biosynthetic pathway was also a confirmed intermediate for L-proline biosynthesis, and its automatic self-cyclization would significantly decrease the efficiency for CPOAA biosynthesis, moreover, the roles of PolN and PolP could not be appropriately assigned in the CPOAA biosynthetic pathway . On the other hand, PolN pertains to GCN5 super family acetyl transferase, which is capable of utilizing broadly flexible substrate as acetyl group acceptor . PolP function is easier to be determined for its significant homology to ArgB, a well characterized protein in L-arginine biosynthetic pathway [11, 13]. Accordingly, it was tentatively proposed that L-glutamate was catalyzed by PolN to give rise to N-acetylglutamate, which was then converted to N- acetylglutamate 5-phosphate in the charge of PolP.
The existence of the demonstrated “pathway redundancy” between the biosynthetic pathways of polyoxin and L-arginine promoted us to ponder the interesting phenomena raised in this study. Since the roles of polN and polP were overlapped with those of argA and argB in L-arginine biosynthesis, it seemed as if PolN and PolP would be redundant and dispensable for polyoxin biosynthesis. However, microbial cells, more specifically, the polyoxin producer, have chronically evolved, and their cell factories should be highly efficient and economical . In this respect, the existence of polN and polP seemed to be reasonable and necessary.
For microbial cells, L-arginine could be synthesized via its biosynthetic pathway; nevertheless, the biosynthesis would be partly decreased once the cells could detect the existence of L-arginine [11, 24]. Put it another way, the switch for L-arginine biosynthesis was not in full turn-on status for microbial cells with consistence of exogenous L-arginine, from the point of view, polN and polP were indispensible for polyoxin biosynthesis. Consequently, mutants of polN or polP were only harbor partial capability of polyoxin production. Another interesting and unexpected phenomenon is the argB mutation in S. cacaoi could not be complemented by natural polP, and further research reveals argB plays essential role for both biosynthesis of polyoxin and L-arginine, namely, argB mutant simultaneously abolishes the phenotypes of growth and polyoxin production, partly addressing the interesting phenotype of CY21. For CY21 mutant, Mutation of argB means the L-arginine biosynthetic pathway was interrupted, with the circumstance occurred, the microbial cells would generate chain stringent responses to turn off unessential secondary metabolisms, such as polyoxin biosynthesis. The unusual and unexpected phenotypes found in this study promote us to explore the precise molecular mechanisms for the impact of L-arginine biosynthesis on regulation of polyoxin production, and the related research is now in progress.
“Pathway redundancy” in metabolic networks was widely distributed in nature, as the representative cases characterized in the biosynthetic pathways of FR008/candicidin  and clavulanic acid . Indeed, the interconnection between different pathways including those of polyoxin and L-arginine suggests that the related proteins should be predominantly responsible for their individual business in the metabolic networks; as a result, “Cross-complementation” should be merely a part-time job.
Five genes (polL-P) in the current research were identified to be involved in CPOAA biosynthesis, and argA and argB in L-arginine biosynthetic pathway contributed to the “cross-complementation” with CPOAA pathway, most interestingly, we found that argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide a case for the research on the “pathway redundancy” in metabolic networks, and lay a solid foundation for target improvement of polyoxin production via synthetic biology strategy.
Materials and Methods
Bacterial strains and plasmids (cosmids)
Bacterial strains and plasmids (cosmids) used in this research are described in Additional file 1: Table S1.
General methods and culture conditions
General approaches for the manipulation of E. coli and Streptomyces were based on the standard methods of Sambrook et al.  or Kieser et al. . Strepmyces were grown on MS agar or in TSB (YEME) liquid medium at 30°C . Liquid fermentation medium (per liter containing: Soy powder 20 g, corn powder 20 g, soluble starch 20 g, glucose 10 g, Yeast extract 10 g, CaCO3 4 g, K2HPO4 2 g, NaNO3 2 g, add tap water till 1 liter) was used for polyoxin production. The MM agar  and M9 medium  were used for detection of growth phenotype for the arg mutant of E. coli or Streptomyces. The final antibiotic concentration used in this study is as follows: ampicillin 100 μg/ml, apramycin 30 μg/ml, kanamycin 50 μg/ml, chloramphenicol 25 μg/ml and thiostrepton 12.5 μg/ml.
DNA sequencing and sequence analysis
DNA sequencing was accomplished at Shanghai Maojor Ltd using Applied Biosystems Model 3730 automated DNA sequencer. Sequence data analysis was performed with the FramePlot online program (http://watson.nih.go.jp/~jun/cgi-bin/frameplot-3.0b.pl) . Sequence homology searches were performed using the NCBI online BLAST software .
Construction of pJTU4620 for mutational analysis of the target pol genes
For construction of pJTU4620, m5A7 cosmid  (containing XbaI and SpeI sites correspondingly located at the both sides of foreign insertion) was initially digested by XbaI, and the resultant cohesive ends were blunted by Klenow Fragment (Fermentas). After that, the blunted linear fragment was self-ligated to render pJTU4619, in which SpeI site was subsequently blocked to generate pJTU4620 with the method described as above.
Independent mutation of polL-polN in pJTU4620 cosmid
For individual mutation of polL-polN in pJTU4620, PCR-targeting technology was performed, and the neo cassettes amplified by different pairs of the primers as described in Additional file 1: Table S2 were independently recombined into pJTU4620 to result in mutation of the target genes, subsequently, the neo cassette was removed from pJTU4620 by XbaI-SpeI double digestion leaving the in frame deletion scar.
Construction and complementation of E.coli argA mutant (thyA and argA double mutant)
For construction of the argA mutant of E. coli, a 3.0-kb fragment containing thyA of E. coli was cloned into pKD46  as negative selective marker, and CH2 (thyA - ) mutant (Additional file 1: Figure S2) was used as starter strain, with primers eargAF1 & eargAR1 and eargAF2 & eargAR2-2, the double arms for argA disruption were independently cloned into pIJ2925 to give pJTU2835, then the EcoRI engineered double arms was cloned into pJTU2183 to give pJTU2836, finally, the aac(3)IV from pJTU2848 was inserted into the KpnI site of pJTU2836 to generate the argA disruption vector, pJTU2847. For the complementation of argA, an EcoRI-NdeI polN fragment from pJTU2930 was cloned into identical sites of pET28a to produce pJTU2838.
Construction and identification of CY7 mutant
For construction of the CY7 mutant, two polP disruption arms were independently amplified with primers (H1L-armF with H1L-armR and H1R-armF with H1R-armR) and cloned in pBlueScriptII SK(+) to form pJTU2814 and pJTU2815, respectively; and then a XbaI-PstI engineered fragment from pJTU2814 was cloned into counterpart sites of pJTU2815 to produce pJTU2816, and the PstI engineered aac(3)IV fragment from pJTU2844 was cloned into pJTU2816 to result in pJTU2845, from which the XbaI-EcoRI engineered fragment was cloned into pHL212 (Tao et al., unpublished) to give the polP disruption vector, pJTU2846. For identification of CY7 mutant, primers H1DIR and H1DIF were used.
Construction, identification and complementation of CY21 and CY22 mutants
For construction of the CY21 mutant, a BglII engineered PCR fragment (primers caargBRf2 and caargBRR) was cloned into BglII-HpaI sites of pOJ446 to generate pJTU4730, and a BglII-XbaI PCR fragment (primers caargB1f and caargBLR2) was inserted into corresponding sites of pJTU4730 to result in pJTU4731. After that, a BglII fragment bearing tsr was cloned into counterpart site of pJTU4731 to form pJTU4731-tsr. PCR with primers caargBef and aegDR was used to identify CY21 mutants. For construction CY22 mutant, polP was further disrupted using CY21 as start strain according to the method described as above. For the complementation of CY21 and CY22, polP and argB were individually cloned into pJTU2170 to form pJTU2870 and pJTU4713.
Construction and complementation of the argB mutant of S. coelicolor A3(2) (CX2)
For construction of the CX2 mutant, the left arm for argB mutation was amplified by KOD-plus (Toyobo) polymerase with primers M145argBLF and M145argBLR, after treated by XbaI, the fragment was cloned into XbaI-EcoRV sites of pOJ260 to give pJTU4709, then the EcoRI-BamHI right arm PCR product amplified with primer M145argBRF M145argBRR was cloned into corresponding sites of pJTU4709 to generate the argB in frame deletion vector, pJTU4710. After that, this vector was conjugated into S. coelicolor A3(2) for construction of argB in frame deletion mutant (CX2) based on the standard protocols . For complementation of argB mutant of S. coelicolor, NdeI-EcoRI fragments containing polP (pJTU2829) and argB (pJTU2883) was individually inserted into pJTU2170 to generate pJTU2870 and pJTU4713.
Purification and assay of polyoxin
Polyoxin produced by S. lividans TK24, S. cacaoi as well as its derivatives was detected by bioassay and LC/MS with Agilent 1100 series LC/MSD Trap system. For the bioassay, Trichosporon cutaneum was used as indicator strain, and the protocol were according to Chen et al. . For the purification and HPLC analysis of polyoxin, the methods were based on Chen at al , and the targeted fraction was collected and condensed before LC/MS analysis.
Conditions for LC/MS analysis
The conditions performed for LC/MS analysis were as follows: Agilent ZORBAX SB-C18 column (4.6 × 250 mm), flow rate 0.3 ml/min at room temperature with elution gradient 5%-40% Methanol: 0.3% TFA (HPLC grade) over 40 min at 0.3 ml/min . The elution was monitored at 262 nm with a DAD detector and the data were analyzed with Agilent data analysis software.
The nucleotide and protein sequences reported in this paper have been deposited in GenBank under the accession number HQ202571.
We are very grateful to Prof. Yi Tang from UCLA for critical reading of the manuscript and valuable support. This work was supported by Grants 973 (2012CB721004) and 863 from the Ministry of Science and Technology, the National Science Foundation of China (31070027, 31270100), the Ministry of Education, the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, the Open Funding Project of the State Key Laboratory of Microbial Metabolism, the Science and Technology Commission of Shanghai Municipality, and Shanghai Leading Academic Discipline Project B203.
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