Systems metabolic engineering of the primary and secondary metabolism of Streptomyces albidoflavus enhances production of the reverse antibiotic nybomycin against multi-resistant Staphylococcus aureus.

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 µg L-1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L-1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3'-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L-1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.

Heterologous Production and Biosynthesis of Threonine-16:0dioic acids with a Hydroxamate Moiety.

Dereplication and genome mining in Streptomyces aureus LU18118 combined with heterologous expression of selected biosynthetic gene clusters (BGCs) led to the discovery of various threonine-16:0dioic acids named lipothrenins. Lipothrenins consist of the core elements l-Thr, d-allo-Thr, or Dhb, which are linked to hexadecanedioic acid by an amide bond. The main compound lipothrenin A (1) carries the N-hydroxylated d-allo form of threonine and expresses a siderophore activity. The lipothrenin BGC was analyzed by a series of deletion experiments. As a result, a variety of interesting genes involved in the recruitment and selective activation of linear 16:0dioic acids, amide bond formation, and the epimerization of l-Thr were revealed. Furthermore, a diiron N-oxygenase was identified that may be directly involved in the monooxygenation of the amide bond. This is divergent from the usual hydroxamate formation mechanism in siderophores, which involves hydroxylation of the free amine prior to amide bond formation. Siderophore activity was observed for all N-hydroxylated lipothrenins by application of the CAS assay method.

Screening of Thiopeptide-Producing Streptomycetes Isolated From the Rhizosphere Soil of Juniperus excelsa.

The identification of an increasing number of drug-resistant pathogens has stimulated the development of new therapeutic agents to combat them. Microbial natural products are among the most important elements when it comes to drug discovery. Today, thiopeptide antibiotics are receiving increasing research attention due to their potent activity against Gram-positive bacteria. In this study, we demonstrated the successful use of a whole-cell microbial biosensor (Streptomyces lividans TK24 pMO16) for the specific detection of thiopeptide antibiotics among the native actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa (Bieb.). Among the native strains, two strains of Streptomyces, namely sp. Je 1-79 and Je 1-613, were identified that were capable of producing thiopeptide antibiotics. A multilocus sequence analysis of five housekeeping genes (gyrB, atpD, recA, rpoB, and trpB) classified them as representatives of two different species of the genus Streptomyces. The thiopeptide antibiotics berninamycin A and B were identified in the extracts of the two strains by means of a dereplication analysis. The berninamycin biosynthetic gene cluster was also detected in the genome of the Streptomyces sp. Je 1-79 strain and showed a high level of similarity (93%) with the ber cluster from S. bernensis. Thus, the use of this whole-cell biosensor during the first stage of the screening process could serve to accelerate the specific detection of thiopeptide antibiotics.

Engineering the precursor pool to modulate the production of pamamycins in the heterologous host S. albus J1074.

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger, induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2.

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

A Promiscuous Halogenase for the Derivatization of Flavonoids.

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.

Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces.

Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNALeu UAA (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms2 i6 A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters.

BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Novel Fredericamycin Variant Overproduced by a Streptomycin-resistant Streptomyces albus subsp. chlorinus Strain.

Streptomycetes are an important source of natural products potentially applicable in the pharmaceutical industry. Many of these drugs are secondary metabolites whose biosynthetic genes are very often poorly expressed under laboratory cultivation conditions. In many cases, antibiotic-resistant mutants exhibit increased production of natural drugs, which facilitates the identification and isolation of new substances. In this study, we report the induction of a type II polyketide synthase gene cluster in the marine strain Streptomyces albus subsp. chlorinus through the selection of streptomycin-resistant mutants, resulting in overproduction of the novel compound fredericamycin C2 (1). Fredericamycin C2 (1) is structurally related to the potent antitumor drug lead fredericamycin A.

Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from Micromonospora endolithica.

Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.

Heterologous production of small molecules in the optimized Streptomyces hosts.

Time span of literature covered: 2010-2018The genome mining of streptomycetes has revealed their great biosynthetic potential to produce novel natural products. One of the most promising exploitation routes of this biosynthetic potential is the refactoring and heterologous expression of corresponding biosynthetic gene clusters in a panel of specifically selected and optimized chassis strains. This article will review selected recent reports on heterologous production of natural products in streptomycetes. In the first part, the importance of heterologous production for drug discovery will be discussed. In the second part, the review will discuss recently developed genetic control elements (such as promoters, ribosome binding sites, terminators) and their application to achieve successful heterologous expression of biosynthetic gene clusters. Finally, the most widely used Streptomyces hosts for heterologous expression of biosynthetic gene clusters will be compared in detail. The article will be of interest to natural product chemists, molecular biologists, pharmacists and all individuals working in the natural products drug discovery field.

Gene ssfg_01967 (miaB) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.

Effect of "ribosome engineering" on the transcription level and production of S. albus indigenous secondary metabolites.

Significant resources are invested into efforts to improve the production yields of natural products from Actinobacteria, a well-recognized source of leads for several industries, most notably pharmaceutical one. Introduction of changes into genes for ribosomal protein S12 (rpsL) and/or 16S rRNA methylation (rsmG) is one of traditional approaches (referred to as ribosomal engineering) towards actinobacterial strain improvement. Yet, true potential of ribosome engineering remains unknown as it is currently coupled to empirical selection for aminoglycoside-resistance; rpsL mutations without such phenotypic expression could not be isolated. Here, we report a systematic and rational ribosome engineering approach to study the effect of a range of rpsL mutations on the production level of different biosynthetic gene clusters (BGC). The severe effect of diverse rpsL mutations together with deletion of rsmG engineered in Streptomyces albus has been revealed on the transcription level of several indigenous BGCs. The aforementioned mutations strongly impacted the transcription of indigenous BGCs, possibly because they alter the transcription of BGC-situated and global regulatory genes. The rsmG deletion with certain rpsL mutations can have a synergistic effect on the transcription level of indigenous BGCs. Our work thus provides the first streptomycete platform for rational engineering and study of virtually any nonlethal rpsL mutation. The tremendous effect of ribosome engineering on the transcription profile of the strains was reported for the first time. A library of described S. albus rpsL*/ΔrsmG strains represents a useful tool for overproducing known secondary metabolites and activating silent biosynthetic gene clusters in Actinobacteria.

Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters.

Natural products are a rich source of potential drugs for many applications. Discovery of natural products through the activation of cryptic gene clusters encoding their biosynthetic pathways, engineering of those biosynthetic pathways and optimization of production yields often rely on the expression of these gene clusters in suitable heterologous host strains. Streptomyces albus J1074 provides high success rates of heterologous cluster expression with high levels of metabolite production, rapid growth and amenability to genetic manipulations. Here, we report the construction of S. albus chassis strains optimized for the discovery of natural products through heterologous expression of secondary metabolite clusters. 15 clusters encoding secondary metabolite biosynthetic pathways were deleted in the chromosome of S. albus Del14. This strain provides a substantially improved compound detection limit, owing to the lack of native secondary metabolites. Furthermore, the production yield of natural products heterologously expressed in S. albus Del14 was higher than in commonly used S. albus J1074 and S. coelicolor hosts. S. albus strains B2P1 and B4 were generated by introduction of additional phage phiC31 attB sites into the chromosome of S. albus Del14, allowing integration of up to four copies of a heterologous gene cluster. Amplification of gene clusters in the chromosome of the constructed strains further improved production yields of the encoded compounds. One cryptic cluster from Streptomyces spp. and two clusters from distantly related Frankia spp. strains were successfully activated in these new chassis strains, leading to the isolation of a new compound fralnimycin.

Heterologous Expression of the Nybomycin Gene Cluster from the Marine Strain Streptomyces albus subsp. chlorinus NRRL B-24108.

Streptomycetes represent an important reservoir of active secondary metabolites with potential applications in the pharmaceutical industry. The gene clusters responsible for their production are often cryptic under laboratory growth conditions. Characterization of these clusters is therefore essential for the discovery of new microbial pharmaceutical drugs. Here, we report the identification of the previously uncharacterized nybomycin gene cluster from the marine actinomycete Streptomyces albus subsp. chlorinus through its heterologous expression. Nybomycin has previously been reported to act against quinolone-resistant Staphylococcus aureus strains harboring a mutated gyrA gene but not against those with intact gyrA. The nybomycin-resistant mutants generated from quinolone-resistant mutants have been reported to be caused by a back-mutation in the gyrA gene that restores susceptibility to quinolones. On the basis of gene function assignment from bioinformatics analysis, we suggest a model for nybomycin biosynthesis.

Native and engineered promoters in natural product discovery.

Covers the period up to 2016Bacterial-based natural products have long represented a promising resource for the development of commercially relevant therapeutics, and more than two thirds of these products have been developed from members of the genus Streptomyces. The extensive sequencing of bacterial genomes suggests that the majority of gene clusters encoding natural products are silent and not expressed under standard laboratory conditions. However, these clusters can be activated through systematic exchanges between native transcriptionally silent promoters and transcriptionally active promoters. Therefore, the availability of well-studied constitutive and inducible promoters is of the utmost importance for identifying natural products encoded by silent gene clusters. This manuscript provides an overview of the promoter control elements for streptomycetes and examples of their successful application in refactoring the biosynthetic pathways of natural products.

Generation of new compounds through unbalanced transcription of landomycin A cluster.

The biosynthetically well-studied landomycin A cluster has been used to demonstrate the unbalancing of gene transcription as an efficient method for the generation of new compounds. Landomycin A structural genes were decoupled from the native regulators LanI and LanK and placed under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. In contrast to their native quantitative and temporal regulation, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article.

Insights into the pamamycin biosynthesis.

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin-producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl-carrier protein (ACP)-encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3-oxoadipyl-CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3-Oxoadipyl-CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate.

Iterative marker excision system.

The deletions of large genomic DNA fragments and consecutive gene knockouts are prerequisites for the generation of organisms with improved properties. One of the key issues in this context is the removal of antibiotic resistance markers from engineered organisms without leaving an active recombinase recognition site. Here, we report the establishment of an iterative marker excision system (IMES) that solves this problem. Based on the phiC31 integrase and its mutant att sites, IMES can be used for highly effective deletion of DNA fragments between inversely oriented B-CC and P-GG sites. The B-CC and P-GG sites are derived from attB and attP by substitution of the central core TT dinucleotide with CC and GG, respectively. An unnatural RR site that resides in the chromosome following deletion is the joining product of the right shoulders of B-CC and P-GG. We show that the RR sites do not recombine with each other as well as the RR site recombines with B-CC. The recombination efficiencies between RR and P-GG or RR and LL are only 0.1 % and 1 %, respectively. Thus, IMES can be used for multistep genomic engineering without risking unwanted DNA recombination. The fabrication of multi-purpose antibiotic cassettes and examples of the utilisation of IMES are described.

Genome rearrangements of Streptomyces albus J1074 lead to the carotenoid gene cluster activation.

Streptomyces albus J1074 is a derivative of the S. albus G1 strain defective in SalG1 restriction-modification system. Genome sequencing of S. albus J1074 revealed that the size of its chromosome is 6.8 Mb with unusually short terminal arms of only 0.3 and 0.4 Mb. Here we present our attempts to evaluate the dispensability of subtelomeric regions of the S. albus J1074 chromosome. A number of large site-directed genomic deletions led to circularization of the S. albus J1074 chromosome and to the overall genome reduction by 307 kb. Two spontaneous mutants with an activated carotenoid cluster were obtained. Genome sequencing and transcriptome analysis indicated that phenotypes of these mutants resulted from the right terminal 0.42 Mb chromosomal region deletion, followed by the carotenoid cluster amplification. Our results indicate that the right terminal 0.42 Mb fragment is dispensable under laboratory conditions. In contrast, the left terminal arm of the S. albus J1074 chromosome contains essential genes and only 42 kb terminal region is proved to be dispensable. We identified overexpressed carotenoid compounds and determined fitness costs of the large genomic rearrangements.