A tRNA modification-based regulatory strategy for Lincomycin biosynthesis in Streptomyces lincolnensis.

In Streptomyces species, the cell cycle involves a switch from an early and vegetative state to a later phase where secondary products including antibiotics are synthesized, aerial hyphae form and sporulation occurs. AdpA, which has two domains, activates the expression of numerous genes involved in the switch from the vegetative growth phase. The adpA mRNA of many Streptomyces species has a UUA codon in a linker region between 5’ sequence encoding one domain and 3’ sequence encoding its other and C-terminal domain. UUA codons are exceptionally rare in Streptomyces, and its functional cognate tRNA is not present in a fully modified and acylated form, in the early and vegetative phase of the cell cycle though it is aminoacylated later. Here, we report candidate recoding signals that may influence decoding of the linker region UUA. Additionally, a short ORF 5’ of the main ORF has been identified with a GUG at, or near, its 5’ end and an in-frame UUA near its 3’ end. The latter is commonly 5 nucleotides 5’ of the main ORF start. Ribosome profiling data show translation of that 5’ region. Ten years ago, UUA-mediated translational bypassing was proposed as a sensor by a Streptomyces phage of its host’s cell cycle stage and an effector of its lytic/lysogeny switch. We provide the first experimental evidence supportive of this proposal.

Microbial co-culture: harnessing intermicrobial signaling for the production of novel antimicrobials.

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.

Streptomyces species: Ideal chassis for natural product discovery and overproduction

There is a real consensus that new antibiotics are urgently needed and are the best chance for combating antibiotic resistance. The phylum Actinobacteria is one of the main producers of new antibiotics, with a recent paradigm shift whereby rare actinomycetes have been increasingly targeted as a source of new secondary metabolites for the discovery of new antibiotics. However, this review shows that the genus Streptomyces is still the largest current producer of new and innovative secondary metabolites. Between January 2015 and December 2020, a significantly high number of novel Streptomyces spp. have been isolated from different environments, including extreme environments, symbionts, terrestrial soils, sediments and also from marine environments, mainly from marine invertebrates and marine sediments. This review highlights 135 new species of Streptomyces during this 6-year period with 108 new species of Streptomyces from the terrestrial environment and 27 new species from marine sources. A brief summary of the different pre-treatment methods used for the successful isolation of some of the new species of Streptomyces is also discussed, as well as the biological activities of the isolated secondary metabolites. A total of 279 new secondary metabolites have been recorded from 121 species of Streptomyces which exhibit diverse biological activity. The greatest number of new secondary metabolites originated from the terrestrial-sourced Streptomyces spp.

Biotechnology advances in β-carotene production by microorganisms

Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus, mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s’ association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.

Polyethylene terephthalate (PET) is one of the most marketed aromatic polyesters in the world with an annual demand in 2022 of approximately 29 million metric tons, expected to increase by 40% by 2030. The escalating volume of PET waste and the current inadequacy of recycling methods have led to an accumulation of PET in the terrestrial ecosystem, thereby posing significant global health risks. The pressing global energy and environmental issues associated with PET underscore the urgent need for “upcycling” technologies. These technologies aim to transform reclaimed PET into higher-value products, addressing both energy concerns and environmental sustainability. Enzyme-mediated biocatalytic depolymerization has emerged as a potentially bio-sustainable method for treating and recycling plastics. Numerous plastic-degrading enzymes have been identified from microbial origins, and advancements in protein engineering have been employed to modify and enhance these enzymes. Microbial metabolic engineering allows for the development of modified microbial chassis capable of degrading PET substrates and converting their derived monomers into industrial relevant products. In this review, we describe several engineering approaches aiming at enhancing the performances of PET-degrading enzymes and we present the current metabolic engineering strategies adopted to bio-upcycle PET into high-value molecules.

Streptomyces albus ( albidoflavus ) J1074 is one of the preferred streptomycete chassis strains for the expression of specialized metabolite biosynthetic gene clusters. Leucyl tRNA gene bldA is one of the regulatory switches that, through delayed translation of its cognate codon UUA, confines the production of specialized metabolites to a stationary phase. An integral step in the maturation of the tRNA UAA is its post-transcriptional tRNA modifications (PTTMs), which are poorly understood. Exploring the installation of BldA PTTMs may reveal their cross-talk with antibiotic biosynthesis regulatory pathways and offer new ways to manipulate specialized metabolism in Streptomyces . In this work, we focused on the J1074 gene XNR_5296 , coding for a SPOUT family tRNA methyltransferase homologous to Escherichia coli TrmL that methylates the ribose residue of uridine (2′-O-methyluridine or Um) at the wobble position of leucyl tRNA UAA . First, we revisited the diversity of modified nucleosides for the wild-type strain and suggest that wobble uridine in tRNA Leu UAA is in the form of s 2 Um. Wobble uridine hypermodifications, such as mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine), cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine), and cmnm 5 Um (5-carboxymethylaminomethyl-2′-O-methyluridine), found in enterobacteria, could not be confirmed for J1074. Second, while an XNR_5296 knockout did not diminish the formation of s 2 Um, it did lead to a strong decrease in the abundance of Um in total nucleoside hydrolyzates. The loss of Um32 in leucyl tRNA GAG , as well as the loss of 2′-O-methylated cytosine 32 (Cm32) in prolyl tRNA GGG , was confirmed by RiboMethSeq profiling of the mutant. Our results are reminiscent of the abrogated TrmJ function responsible for position 32 C/U methylation in Gram-negative bacteria. Notably, our findings are the first demonstration of TrmJ-controlled methylation in Gram-positive bacteria. This work expands the understanding of tRNA modification systems in streptomycetes and their potential impact on specialized metabolite production. IMPORTANCE Post-transcriptional modifications are ubiquitous in tRNAs, where they play important structural and regulatory roles. As the types of modified nucleosides and their genetic control differ even between closely related bacterial taxa, there is a need to study them across the entire phylogenetic tree. We recently initiated studies of genetics and chemistry of tRNA modifications in streptomycetes, one of the most prolific producers of specialized metabolites of immense practical value (antibiotics, anticancer drugs, to name just a few). A point of special interest was the modifications of leucyl tRNA UAA , the only one capable of decoding the rarest in Streptomyces codon UUA. In a search for a TrmL homologue responsible for 2′-O-methylation of the wobble nucleoside 34 (U) ribose of tRNA UAA , we probed the function of gene XNR_5296 . XNR_5296 knockout led to the loss of 2′-O-methylated uridine 32 (Um) in leucyl tRNA GAG and 2′-O-methylated cytosine 32 (Cm) in prolyl tRNA GGG . This result, as well as in silico analysis, suggests parallels between Xnr_5296 and the Escherichia coli TrmJ enzyme responsible for U/C methylation at position 32 of glutaminyl tRNA UUG and tRNA CUG , methionyl tRNA CAU , seryl tRNA UGA , and tryptophanyl tRNA CCA , although the Streptomyces counterpart methylates different tRNA species. Thus, our work reveals previously unreported tRNA modification and its gene in Streptomyces and serves as a stepping stone to further interrogate the functions of highly paralogous SPOUT family methyltransferases in this important bacterial genus.

Streptomyces species are soil-dwelling bacteria that produce vast numbers of pharmaceutically valuable secondary metabolites (SMs), such as antibiotics, immunosuppressants, antiviral, and anticancer drugs. On the other hand, the biosynthesis of most SMs remains very low due to tightly controlled regulatory networks. Both global and pathway-specific regulators are involved in the regulation of a specific SM biosynthesis in various Streptomyces species. Over the past few decades, many of these regulators have been identified and new ones are still being discovered. Among them, a global regulator of SM biosynthesis named WblA was identified in several Streptomyces species. The identification and understanding of the WblAs have greatly contributed to increasing the productivity of several Streptomyces SMs. This review summarizes the characteristics and applications on WblAs reported to date, which were found in various Streptomyces species and other actinobacteria.

BackgroundThe robustness of Saccharomyces cerevisiae in facilitating industrial-scale production of ethanol extends its utilization as a platform to synthesize other metabolites. Metabolic engineering strategies, typically via pathway overexpression and deletion, continue to play a key role for optimizing the conversion efficiency of substrates into the desired products. However, chemical production titer or yield remains difficult to predict based on reaction stoichiometry and mass balance. We sampled a large space of data of chemical production from S. cerevisiae, and developed a statistics-based model to calculate production yield using input variables that represent the number of enzymatic steps in the key biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability.ResultsBased on the production data of about 40 chemicals produced from S. cerevisiae, metabolic engineering methods, nutrient supplementation, and fermentation conditions described therein, we generated mathematical models with numerical and categorical variables to predict production yield. Statistically, the models showed that: 1. Chemical production from central metabolic precursors decreased exponentially with increasing number of enzymatic steps for biosynthesis (>30% loss of yield per enzymatic step, P-value = 0); 2. Categorical variables of gene overexpression and knockout improved product yield by 2~4 folds (P-value < 0.1); 3. Addition of notable amount of intermediate precursors or nutrients improved product yield by over five folds (P-value < 0.05); 4. Performing the cultivation in a well-controlled bioreactor enhanced the yield of product by three folds (P-value < 0.05); 5. Contribution of oxygen to product yield was not statistically significant. Yield calculations for various chemicals using the linear model were in fairly good agreement with the experimental values. The model generally underestimated the ethanol production as compared to other chemicals, which supported the notion that the metabolism of Saccharomyces cerevisiae has historically evolved for robust alcohol fermentation.ConclusionsWe generated simple mathematical models for first-order approximation of chemical production yield from S. cerevisiae. These linear models provide empirical insights to the effects of strain engineering and cultivation conditions toward biosynthetic efficiency. These models may not only provide guidelines for metabolic engineers to synthesize desired products, but also be useful to compare the biosynthesis performance among different research papers.

Streptomycetes are well-known producers of biologically active secondary metabolites. Various efforts have been made to increase productions of these metabolites, while few approaches could well coordinate the biosynthesis of secondary metabolites and other physiological events of their hosts. Here we develop a universal autoregulated strategy for fine-tuning the expression of secondary metabolites biosynthetic gene clusters (BGCs) in Streptomyces species. First, inducible promoters were used to control the expression of secondary metabolites BGCs. Then, the optimal induction condition was determined by response surface model in both dimensions of time and strength. Finally, native promoters with similar transcription profile to the inducible promoter under the optimal condition were identified based on time-course transcriptome analyses, and used to replace the inducible promoter following an elaborate replacement approach. The expression of actinorhodin (Act) and heterogeneous oxytetracycline (OTC) BGCs were optimized in Streptomyces coelicolor using this strategy. Compared to modulating the expression via constitutive promoters, our strategy could dramatically improve the titers of Act and OTC by 1.3- and 9.1-fold, respectively. The autoregulated fine-tuning strategy developed here opens a novel route for titer improvement of desired secondary metabolites in Streptomyces.

EDITORIAL article Front. Microbiol., 11 January 2016Sec. Microbial Physiology and Metabolism Volume 6 - 2015 | https://doi.org/10.3389/fmicb.2015.01562

Streptomyces species are well known for their particular features of morphological differentiation. On solid agar, a mold-like aerial mycelium is formed and spores are produced, in which the bld genes play a crucial role. In S. coelicolor, mutations in one specific bld gene called bldA led to a "naked" phenotype lacking aerial hyphae and spores. This peculiar behavior became a major interest for scientific research in the past and it was revealed that bldA is coding for a unique tRNA able to translate a UUA codon into the amino acid leucine. UUA codons are a very rare property of G + C-rich Streptomyces genomes. The impact of bldA on morphology can in parts be attributed to the regulatory effect of bldA on the translational level, because TTA-containing genes can only be translated into their corresponding protein in the presence of a fully functioning bldA gene. In addition to the visible effect of bldA expression on the phenotype of S. coelicolor, bldA mutants were also deficient in antibiotic production. This led to the assumption that the role of bldA must exceed translational control. Many TTA-containing genes are coding for transcriptional regulators which are activating or repressing the transcription of many more genes. Proteomics and transcriptomics are two powerful methods for identifying bldA target genes and it was possible to assign also post-translational regulation to bldA. This review wants to give a short overview on the importance of bldA as a regulator of morphological differentiation and antibiotic production by switching on "silent" gene clusters in Streptomyces.

Metabolic engineering strategies toward production of biofuels