Rongfeng Li | Johns Hopkins University (original) (raw)
Papers by Rongfeng Li
Chemical Science
A small library of stereochemically-defined α,β-diamino acids has been prepared to generate non-n... more A small library of stereochemically-defined α,β-diamino acids has been prepared to generate non-native monobactams through fermentation, highlighting the potential of this platform for directed biosynthesis.
Chemical Communications, 2019
TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor t... more TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor three or two times, respectively, in a sequential, non-processive fashion.
Nature chemical biology, 2018
The N-sulfonated monocyclic β-lactam ring characteristic of the monobactams confers resistance to... more The N-sulfonated monocyclic β-lactam ring characteristic of the monobactams confers resistance to zinc metallo-β-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented nonribosomal peptide synthetase activities, wherein an assembled tripeptide is N-sulfonated in trans before direct synthesis of the β-lactam ring in a noncanonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.
ACS chemical biology, Oct 22, 2017
We have produced draft whole-genome sequences for two bacterial strains reported to produce the b... more We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose reclassification of ATCC 31363 from the original Pseudomonas genus to Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia mesoacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin pathway in both P. acidophila and B. mesoacidophila are likely involved in bulgecin A biosynthesis.
Proceedings of the National Academy of Sciences, 1998
The principal cause of bacterial resistance to penicillin and other β-lactam antibiotics is the a... more The principal cause of bacterial resistance to penicillin and other β-lactam antibiotics is the acquisition of plasmid-encoded β-lactamases, enzymes that catalyze hydrolysis of the β-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of β-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical β-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg 2+ -dependent enzyme, a β-lactam synthetase (β-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N 2 -(carboxyethyl)- l -arginine (CEA). Chemica...
Proceedings of the National Academy of Sciences, 2008
The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyce... more The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the β-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF , was shown to encode a protein capable of N -acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with...
The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infection... more The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infections that are problematic to treat due to their metallo-b-lactamase mediated resistance against conventional b-lactam antibiotics. Herein, we report a divergent synthesis of C3-substituted 2,3-diaminopropionates featuring an array of small functional groups and examine their potential as alternative precursors during monobactam biosynthesis in a mutant strain (DsulG) of Pseudomonas acidophila that is deficient in the supply of this native precursor. In vitro assays revealed high diastereoselectivity, as well as a substrate tolerance by the terminal adenylation domain of the non-ribosomal peptide synthetase (NRPS) SulM toward the majority of synthetic analogs. Chemical complementation of this mutant yielded a fluorinated, bioactive monobactam through fermentation as confirmed by a combination of spectrometric data and microbiological assays. This study demonstrates site-specific functionalization of a clinically important natural product and sets in place a platform for further strain improvements and engineered NRPS-biosynthesis of non-native congeners.
Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. The... more Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections.
Their biosynthetic gene clusters contain three essential B12-dependent radical
S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this
subfamily catalyze methyl transfer, but only one is required to sequentially install all
methionine-derived carbons in complex carbapenems. Therefore, it is probable that the
other two rSAMenzymes have noncanonical functions. Through a series of fermentation
and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether
bond formation between C2 of a carbapenam precursor and pantetheine, uniting
initial bicycle assembly common to all carbapenems with later tailoring events unique to
complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine.
Neither of these functions has been observed previously in a B12-dependent radical
SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond
carbon–carbon bond formation or rearrangement. It is also the only radical SAM
enzyme currently known to catalyze carbon–sulfur bond formation with only an rSAM
Fe–S cluster and no additional auxiliary clusters.
Complex carbapenem b-lactam antibiotics contain diverse C6 alkyl substituents constructed by coba... more Complex carbapenem b-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes. TokK installs the C6 isopropyl chain found in asparenomycin. Time-course analyses of TokK and its ortholog ThnK, which forms the C6 ethyl chain of thienamycin, indicate that catalysis occurs through a sequence of discrete, non-processive methyl transfers.
The N-sulfonated monocyclic b-lactam ring characteristic of the monobactams confers resistance t... more The N-sulfonated monocyclic b-lactam ring characteristic
of the monobactams confers resistance to zinc metallo-blactamases
and affords the most effective class to combat
carbapenem-resistant enterobacteria (CRE). Here we report
unprecedented nonribosomal peptide synthetase activities,
wherein an assembled tripeptide is N-sulfonated in trans
before direct synthesis of the b-lactam ring in a noncanonical,
cysteine-containing thioesterase domain. This means of
azetidinone synthesis is distinct from the three others known
in nature.
Highlights d The first monobactam biosynthetic gene cluster is isolated and characterized d Two n... more Highlights d The first monobactam biosynthetic gene cluster is isolated and characterized d Two non-ribosomal peptide synthetases play essential biosynthetic roles d A new mechanism of b-lactam formation is catalyzed in an aberrant thioesterase domain
We have produced draft whole-genome sequences for two bacterial strains reported to produce the b... more We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.
Metabolic Engineering, 2006
Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and ... more Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.
The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyce... more The enzymatic activities of three proteins encoded by the thienamycin
gene cluster of Streptomyces cattleya (ThnR, ThnH, and
ThnT) have been shown to incrementally cleave CoA to afford the
active side-chain component of the -lactam antibiotic thienamycin.
These results supersede proposals based on earlier radiochemical
incorporation experiments. For 20 years it has been thought
that cysteine was directly incorporated into the antibiotic. Specific,
stepwise truncation of CoA to 4-phosphopantetheine, pantetheine,
and finally cysteamine was observed with ThnR, ThnH, and
ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated
carbapenams were synthesized to address possible
thienamycin biosynthetic intermediates and were shown to be
effective substrates for the pantetheine-cleaving enzyme ThnT.
Finally, a fourth gene, thnF, was shown to encode a protein capable
of N-acetylating a model compound containing cysteamine in the
presence of acetyl-CoA, consistent with the production of the S.
cattleya cometabolite, N-acetylthienamycin. Taken together, these
four enzymes are proposed to siphon CoA from primary metabolism
to create the side chains for the predominant S. cattleya
carbapenems, thienamycin and N-acetylthienamycin, in a process
likely to be general for the broader class of these antibiotics.
Clavulanic acid is a potent inhibitor of -lactamase enzymes and is of demonstrated value in the ... more Clavulanic acid is a potent inhibitor of -lactamase enzymes and is of demonstrated value in the treatment of infections by -lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219-236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.
ChemBioChem, 2014
Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, hav... more Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C-2 and C-6 side chains as well as the stereochemistry at C-5/C-6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. While the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by S. argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that genes encoding the biosynthesis of the bicyclic core and the C-6 and C-2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22/23, encode a twocomponent response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.
(5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring c... more (5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem-lactam antibiotics. It co-occurs with two saturated (3S,5S)and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the "inverted" (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on R-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, R-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class.
Carbapenem-3-carboxylic acid (4) is the simplest structurally of over 60 naturally occurring carb... more Carbapenem-3-carboxylic acid (4) is the simplest structurally of over 60 naturally occurring carbapenem-lactam antibiotics isolated since the discovery of thienamycin, 1 whose gene cluster has been recently identified. 2 Members of this family and their derivatives are clinically important for their broad spectrum of antibiotic activity and their relative resistance to most clinically encountered-lactamases. 3 We determined that the assembly of all three-lactams 2, 3, and 4 produced by Erwinia carotoVora (now Pectobacterium carotoVorum) 2 and Serratia sp. ATCC 39006 is carried out through the action of just three enzymes, CarA (carbapenam synthetase), CarB, and CarC (Scheme 1). 4-6 This process is accompanied by a remarkable stereochemical inversion at C-5 of 2 to both 3 and 4, which proceeds by loss of hydrogen label at that stereocenter catalyzed by CarC (carbapenem synthase). 6
The invention methods for the enhancement of clavulanic acid production. In particular, the inven... more The invention methods for the enhancement of clavulanic acid production. In particular, the invention provides a method for increasing the production of clavulanic acid by: gene dosage with orf2 from the clavulanic acid biosynthetic pathway in Streptomyces clavuligerus; and by manipulation of fermentation conditions, especially the concentration of DG3P, a substrate of N2(carboxyethyl)arginine synthase, the protein encoded by orf2. A method for preparing N2(carboxyethyl)arginine synthase is also provided, as is an assay for identifying its substrates.
Chemical Science
A small library of stereochemically-defined α,β-diamino acids has been prepared to generate non-n... more A small library of stereochemically-defined α,β-diamino acids has been prepared to generate non-native monobactams through fermentation, highlighting the potential of this platform for directed biosynthesis.
Chemical Communications, 2019
TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor t... more TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor three or two times, respectively, in a sequential, non-processive fashion.
Nature chemical biology, 2018
The N-sulfonated monocyclic β-lactam ring characteristic of the monobactams confers resistance to... more The N-sulfonated monocyclic β-lactam ring characteristic of the monobactams confers resistance to zinc metallo-β-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented nonribosomal peptide synthetase activities, wherein an assembled tripeptide is N-sulfonated in trans before direct synthesis of the β-lactam ring in a noncanonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.
ACS chemical biology, Oct 22, 2017
We have produced draft whole-genome sequences for two bacterial strains reported to produce the b... more We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose reclassification of ATCC 31363 from the original Pseudomonas genus to Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia mesoacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin pathway in both P. acidophila and B. mesoacidophila are likely involved in bulgecin A biosynthesis.
Proceedings of the National Academy of Sciences, 1998
The principal cause of bacterial resistance to penicillin and other β-lactam antibiotics is the a... more The principal cause of bacterial resistance to penicillin and other β-lactam antibiotics is the acquisition of plasmid-encoded β-lactamases, enzymes that catalyze hydrolysis of the β-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of β-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical β-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg 2+ -dependent enzyme, a β-lactam synthetase (β-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N 2 -(carboxyethyl)- l -arginine (CEA). Chemica...
Proceedings of the National Academy of Sciences, 2008
The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyce... more The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the β-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF , was shown to encode a protein capable of N -acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with...
The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infection... more The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infections that are problematic to treat due to their metallo-b-lactamase mediated resistance against conventional b-lactam antibiotics. Herein, we report a divergent synthesis of C3-substituted 2,3-diaminopropionates featuring an array of small functional groups and examine their potential as alternative precursors during monobactam biosynthesis in a mutant strain (DsulG) of Pseudomonas acidophila that is deficient in the supply of this native precursor. In vitro assays revealed high diastereoselectivity, as well as a substrate tolerance by the terminal adenylation domain of the non-ribosomal peptide synthetase (NRPS) SulM toward the majority of synthetic analogs. Chemical complementation of this mutant yielded a fluorinated, bioactive monobactam through fermentation as confirmed by a combination of spectrometric data and microbiological assays. This study demonstrates site-specific functionalization of a clinically important natural product and sets in place a platform for further strain improvements and engineered NRPS-biosynthesis of non-native congeners.
Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. The... more Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections.
Their biosynthetic gene clusters contain three essential B12-dependent radical
S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this
subfamily catalyze methyl transfer, but only one is required to sequentially install all
methionine-derived carbons in complex carbapenems. Therefore, it is probable that the
other two rSAMenzymes have noncanonical functions. Through a series of fermentation
and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether
bond formation between C2 of a carbapenam precursor and pantetheine, uniting
initial bicycle assembly common to all carbapenems with later tailoring events unique to
complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine.
Neither of these functions has been observed previously in a B12-dependent radical
SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond
carbon–carbon bond formation or rearrangement. It is also the only radical SAM
enzyme currently known to catalyze carbon–sulfur bond formation with only an rSAM
Fe–S cluster and no additional auxiliary clusters.
Complex carbapenem b-lactam antibiotics contain diverse C6 alkyl substituents constructed by coba... more Complex carbapenem b-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes. TokK installs the C6 isopropyl chain found in asparenomycin. Time-course analyses of TokK and its ortholog ThnK, which forms the C6 ethyl chain of thienamycin, indicate that catalysis occurs through a sequence of discrete, non-processive methyl transfers.
The N-sulfonated monocyclic b-lactam ring characteristic of the monobactams confers resistance t... more The N-sulfonated monocyclic b-lactam ring characteristic
of the monobactams confers resistance to zinc metallo-blactamases
and affords the most effective class to combat
carbapenem-resistant enterobacteria (CRE). Here we report
unprecedented nonribosomal peptide synthetase activities,
wherein an assembled tripeptide is N-sulfonated in trans
before direct synthesis of the b-lactam ring in a noncanonical,
cysteine-containing thioesterase domain. This means of
azetidinone synthesis is distinct from the three others known
in nature.
Highlights d The first monobactam biosynthetic gene cluster is isolated and characterized d Two n... more Highlights d The first monobactam biosynthetic gene cluster is isolated and characterized d Two non-ribosomal peptide synthetases play essential biosynthetic roles d A new mechanism of b-lactam formation is catalyzed in an aberrant thioesterase domain
We have produced draft whole-genome sequences for two bacterial strains reported to produce the b... more We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam β-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.
Metabolic Engineering, 2006
Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and ... more Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.
The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyce... more The enzymatic activities of three proteins encoded by the thienamycin
gene cluster of Streptomyces cattleya (ThnR, ThnH, and
ThnT) have been shown to incrementally cleave CoA to afford the
active side-chain component of the -lactam antibiotic thienamycin.
These results supersede proposals based on earlier radiochemical
incorporation experiments. For 20 years it has been thought
that cysteine was directly incorporated into the antibiotic. Specific,
stepwise truncation of CoA to 4-phosphopantetheine, pantetheine,
and finally cysteamine was observed with ThnR, ThnH, and
ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated
carbapenams were synthesized to address possible
thienamycin biosynthetic intermediates and were shown to be
effective substrates for the pantetheine-cleaving enzyme ThnT.
Finally, a fourth gene, thnF, was shown to encode a protein capable
of N-acetylating a model compound containing cysteamine in the
presence of acetyl-CoA, consistent with the production of the S.
cattleya cometabolite, N-acetylthienamycin. Taken together, these
four enzymes are proposed to siphon CoA from primary metabolism
to create the side chains for the predominant S. cattleya
carbapenems, thienamycin and N-acetylthienamycin, in a process
likely to be general for the broader class of these antibiotics.
Clavulanic acid is a potent inhibitor of -lactamase enzymes and is of demonstrated value in the ... more Clavulanic acid is a potent inhibitor of -lactamase enzymes and is of demonstrated value in the treatment of infections by -lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219-236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.
ChemBioChem, 2014
Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, hav... more Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C-2 and C-6 side chains as well as the stereochemistry at C-5/C-6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. While the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by S. argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that genes encoding the biosynthesis of the bicyclic core and the C-6 and C-2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22/23, encode a twocomponent response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.
(5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring c... more (5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem-lactam antibiotics. It co-occurs with two saturated (3S,5S)and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the "inverted" (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on R-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, R-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class.
Carbapenem-3-carboxylic acid (4) is the simplest structurally of over 60 naturally occurring carb... more Carbapenem-3-carboxylic acid (4) is the simplest structurally of over 60 naturally occurring carbapenem-lactam antibiotics isolated since the discovery of thienamycin, 1 whose gene cluster has been recently identified. 2 Members of this family and their derivatives are clinically important for their broad spectrum of antibiotic activity and their relative resistance to most clinically encountered-lactamases. 3 We determined that the assembly of all three-lactams 2, 3, and 4 produced by Erwinia carotoVora (now Pectobacterium carotoVorum) 2 and Serratia sp. ATCC 39006 is carried out through the action of just three enzymes, CarA (carbapenam synthetase), CarB, and CarC (Scheme 1). 4-6 This process is accompanied by a remarkable stereochemical inversion at C-5 of 2 to both 3 and 4, which proceeds by loss of hydrogen label at that stereocenter catalyzed by CarC (carbapenem synthase). 6
The invention methods for the enhancement of clavulanic acid production. In particular, the inven... more The invention methods for the enhancement of clavulanic acid production. In particular, the invention provides a method for increasing the production of clavulanic acid by: gene dosage with orf2 from the clavulanic acid biosynthetic pathway in Streptomyces clavuligerus; and by manipulation of fermentation conditions, especially the concentration of DG3P, a substrate of N2(carboxyethyl)arginine synthase, the protein encoded by orf2. A method for preparing N2(carboxyethyl)arginine synthase is also provided, as is an assay for identifying its substrates.