Mediterranei U32 Research Articles

CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.

GlnR positive transcriptional regulation of the phosphate-specific transport system pstSCAB in Amycolatopsis mediterranei U32.

Amycolatopsis mediterranei U32 is an important industrial strain for the production of rifamycin SV. Rifampicin, a derivative of rifamycin SV, is commonly used to treat mycobacterial infections. Although phosphate has long been known to affect rifamycin biosynthesis, phosphate transport, metabolism, and regulation are poorly understood in A. mediterranei. In this study, the functional phosphate transport system pstSCAB was isolated by RNA sequencing and inactivated by insertion mutation in A. mediterranei U32. The mycelium morphology changed from a filamentous shape in the wild-type and pstS1+ strains to irregular swollen shape at the end of filamentous in the ΔpstS1 strain. RT-PCR assay revealed that pstSCAB genes are co-transcribed as a polycistronic messenger. The pstSCAB transcription was significantly activated by nitrate supplementation and positively regulated by GlnR which is a global regulator of nitrogen metabolism in actinomycetes. At the same time, the yield of rifamycin SV decreased after mutation (ΔpstS1) compared with wild-type U32, which indicated a strong connection among phosphate metabolism, nitrogen metabolism, and rifamycin production in actinomycetes.

Conjugation of φBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32

ABSTRACT The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 × 10−5 CFU per recipient cell. This integrative vector, based on the φBT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.

Two genes, <italic>rif15</italic> and <italic>rif16</italic>, of the rifamycin biosynthetic gene cluster in <italic>Amycolatopsis mediterranei</italic> likely encode a transketolase and a P450 monooxygenase, respectively, both essential for the conversion of rifamycin SV into B

Amycolatopsis mediterranei produces an important antibiotic rifamycin, the biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B. In this study, we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was able to convert rifamycin SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, but not in Streptomyce coelicolor or Mycobacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded by the rif cluster should be involved in this process.

Bacterial Type I Glutamine Synthetase of the Rifamycin SV Producing Actinomycete, <italic>Amycolatopsis mediterranei</italic> U32, is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.

Cloning and Preliminary Characterization of lh3 Gene Encoding a Putative Acetyltransferase from a Rifamycin SV-Producing Strain Amycolatopsis mediterranei

An ORF located immediately downstream of glnR gene was cloned from Amycolatopsis mediterranei U32 and was named lh3. Sequence analysis revealed that lh3 encodes a putative acetyltransferase, which shows high amino acid sequence similarities to the mycothiol synthase (MshD) from other actinomycetes. For functional analysis, mutation in lh3 gene was generated by gene replacement with an apramycin resistance gene through homologous recombination. Compared with the wild type strain, the resulting mutant was more sensitive to H2O2, apramycin and erythromycin by two- to three-fold. These results suggest that the lh3 gene plays an important role in the course of detoxification in A. mediterranei U32.

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins ( oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.

A novel two-component system amrB-amkB involved in the regulation of central carbohydrate metabolism in rifamycin SV-producing Amycolatopsis mediterranei U32.

A novel two-component signal transduction system amrB-amkB was cloned from rifamycin SV-producing Amycolatopsis mediterranei U32, and their biochemical functions as a response regulator and a histidine protein kinase, respectively, were proven. The amrB disruption mutant was generated by insertional inactivation with the aparmycin resistance gene. The metabolic response to the absence of amrB gene was determined by a biochemical profiling technique in which the concentration changes of metabolic intermediates were measured by gas chromatography with time-of-flight mass spectrometry (GC/TOF-MS). Although the phenotype analyses of the amrB gene disruption mutant showed no significant change with respect to rifamycin SV production and morphological differentiation, the global metabolomic analyses found the concentration levels of some key intermediates in the TCA cycle and glycolysis pathway were affected by an amrB gene disruption event. The primary results suggested that amrB-amkB genes might be involved in the regulation of central carbohydrate metabolism in A. mediterranei U32.

MoeA, an enzyme in the molybdopterin synthesis pathway, is required for rifamycin SV production in Amycolatopsis mediterranei U32.

Rifamycin SV contains one amide nitrogen atom at its C(7)N moiety. Earlier labeling studies suggested that nitrogen might be incorporated from a pathway involved in a molybdenum-dependent nitrate reductase. However, no genetic evidence is available thus far. The structural gene moeA, which is involved in molybdopterin synthesis in various organisms, has been cloned from rifamycin SV-producing Amycolatopsis mediterranei strain U32. The amino acid sequence deduced from the moeA gene showed significant similarity to members of the MoeA protein family and contains all the structural features that are highly conserved in the putative functional domains of MoeA proteins. Southern hybridization showed that there is only one moeA gene in the A. mediterranei genome. To further investigate the possible physiological function of the moeA gene, a double crossover gene replacement was achieved by inserting an aparmycin resistance gene into moeA in the A. mediterranei U32 chromosome. Phenotype analysis showed that the moeA gene is required for A. mediterranei growth in a minimal medium with nitrate as sole nitrogen source, possibly because nitrate reductase activity is diminished due to disruption of the moeA gene. Compared to the wild type strain, moeA-disrupted mutants lost 95% of their rifamycin SV production capacity in complex fermentation media. The results demonstrate that the moeA gene is necessary for rifamycin SV production in A. mediterranei, and that the nitrogen assimilation pathway involved in nitrate reductase is the major pathway for the genesis of the amide nitrogen atom in the rifamycin SV molecule.

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.

An Eukaryotic-Type Serine/Threonine Protein Kinase Involved in the Carbon Source-Dependent Pigment Biosynthesis in Amycolatopsis mediterranei U32

The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases. It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases. The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis. The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. mediterranei chromosomal DNA. The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A. mediterranei U32.

Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32.

The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E. coli X6118 (asd-), respectively. The A. mediterranei U32 asparto-kinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine. Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A. mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.

Molecular analysis and heterologous expression of the gene encoding methylmalonyl-coenzyme A mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32.

The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV-producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonesis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mutAB gene, was quite similar to the ORFs downstream of mutAB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B12. A purification procedure based on the B12 affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%.