Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin.

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 µg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

Isolation of depsipeptides and optimization for enhanced production of valinomycin from the North-Western Himalayan cold desert strain Streptomyces lavendulae.

Exploration of microbial dynamics of Streptomyces lavendulae ACR-DA1, a psychrotrophic isolate from the North-Western Himalayan cold desert, was carried out using matrix-assisted laser desorbtion ionisation-time of flight mass spectrometer. Valinomycin was found as a major produce and cyclic depsipeptide montanastatin as a minor produce. The yield of the valinomycin was found to be 0.3 mg l-1 in submerged growth condition at the batch scale. Miniaturization of optimization experiments was adept to maximize the production using the expeditious and efficient technique of intact cell mass spectrometry. The present study showed that using optimized conditions and growing the culture in synthetic mineral base starch medium at 10 °C enhanced the production to 19.4 mg l-1. Our results demonstrated 64-fold increase in yield from the wild-type S. lavendulae ACR-DA1 strain using a simple and economical downstream process.

Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid.

Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates1. These enzymes include serine hydrolases2,3 (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery4,5. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours6. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight7-10, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code11. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase12, we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.

Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions.

Streptomyces species are amongst the most exploited microorganisms due to their ability to produce a plethora of secondary metabolites with bioactive potential, including several well known drugs. They are endowed with immense unexplored potential and substantial efforts are required for their isolation as well as characterization for their bioactive potential. Unexplored niches and extreme environments are host to diverse microbial species. In this study, we report Streptomyces lavendulae ACR-DA1, isolated from extreme cold deserts of the North Western Himalayas, which produces a macrolactone antibiotic, valinomycin. Valinomycin is a K+ ionophoric non-ribosomal cyclodepsipeptide with a broad range of bioactivities including antibacterial, antifungal, antiviral and cytotoxic/anticancer activities. Production of valinomycin by the strain S. lavendulae ACR-DA1 was studied under different fermentation conditions like fermentation medium, temperature and addition of biosynthetic precursors. Synthetic medium at 10°C in the presence of precursors i.e. valine and pyruvate showed enhanced valinomycin production. In order to assess the impact of various elicitors, expression of the two genes viz. vlm1 and vlm2 that encode components of heterodimeric valinomycin synthetase, was analyzed using RT-PCR and correlated with quantity of valinomycin using LC-MS/MS. Annelid, bacterial and yeast elicitors increased valinomycin production whereas addition of fungal and plant elicitors down regulated the biosynthetic genes and reduced valinomycin production. This study is also the first report of valinomycin biosynthesis by Streptomyces lavendulae.

Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression.

Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations.

BACKGROUND: Heterologous production of natural products in Escherichia coli has emerged as an attractive strategy to obtain molecules of interest. Although technically feasible most of them are still constrained to laboratory scale production. Therefore, it is necessary to develop reasonable scale-up strategies for bioprocesses aiming at the overproduction of targeted natural products under industrial scale conditions. To this end, we used the production of the antibiotic valinomycin in E. coli as a model system for scalable bioprocess development based on consistent fed-batch cultivations. RESULTS: In this work, the glucose limited fed-batch strategy based on pure mineral salt medium was used throughout all scales for valinomycin production. The optimal glucose feed rate was initially detected by the use of a biocatalytically controlled glucose release (EnBase® technology) in parallel cultivations in 24-well plates with continuous monitoring of pH and dissolved oxygen. These results were confirmed in shake flasks, where the accumulation of valinomycin was highest when the specific growth rate decreased below 0.1 h(-1). This correlation was also observed for high cell density fed-batch cultivations in a lab-scale bioreactor. The bioreactor fermentation produced valinomycin with titers of more than 2 mg L(-1) based on the feeding of a concentrated glucose solution. Valinomycin production was not affected by oscillating conditions (i.e. glucose and oxygen) in a scale-down two-compartment reactor, which could mimic similar situations in industrial bioreactors, suggesting that the process is very robust and a scaling of the process to a larger industrial scale appears a realistic scenario. CONCLUSIONS: Valinomycin production was scaled up from mL volumes to 10 L with consistent use of the fed-batch technology. This work presents a robust and reliable approach for scalable bioprocess development and represents an example for the consistent development of a process for a heterologously expressed natural product towards the industrial scale.

Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation.

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L⁻¹ by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L⁻¹. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD600>50 and a valinomycin titer of appr. 10 mg L⁻¹. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.

Reconstituted biosynthesis of the nonribosomal macrolactone antibiotic valinomycin in Escherichia coli.

The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous expression of NRPSs in a robust and easy to manipulate expression host is an attractive strategy to make pharmaceutically relevant NRPs more accessible and is also a basis for engineering of these enzymes to generate novel synthetic bioactive compounds. Here we show a systematic approach for the heterologous expression of the 654 kDa heterodimeric valinomycin synthetase (VlmSyn) from Streptomyces tsusimaensis in a soluble and active form in Escherichia coli. VlmSyn activity and precursor requirements were determined in vitro and provided evidence for a previously proposed model of valinomycin biosynthesis. In vivo production of recombinant valinomycin, a macrolactone antibiotic with reported antifungal, antibacterial, and antiviral activities, was achieved using an engineered E. coli strain growing in inexpensive media and independent of the supplementation with precursors and further optimization of the cultivation conditions. Tailoring of VlmSyn in E. coli paves the way to the production of novel valinomycin analogues in the future.

Cereulide and valinomycin, two important natural dodecadepsipeptides with ionophoretic activities.

Cereulide produced by Bacillus cereus sensu stricto and valinomycin synthesized mainly by Streptomyces spp. are natural dodecadepsipeptide ionophores that act as potassium transporters. Moreover, they comprise three repetitions of similar tetrapeptide motifs synthesized by nonribosomal peptide synthesis complexes. Resemblances in their structure find their reflections in the same way of action. The toxicity of valinomycin and cereulide is an effect of the disturbance of ionic equilibrium and transmembrane potential that may influence the whole organism and then cause fatal consequences. The vlm and ces operons encoding valinomycin and cereulide are both composed of two large, similar synthetase genes, one thioestrase gene and four other ORFs with unknown activities. In spite of the characterization of valinomycin and cereulide, genetic determinants encoding their biosynthesis have not yet been clarified.

Deciphering the biosynthetic codes for the potent anti-SARS-CoV cyclodepsipeptide valinomycin in Streptomyces tsusimaensis ATCC 15141.

Valinomycin was recently reported to be the most potent agent against severe acute respiratory-syndrome coronavirus (SARS-CoV) in infected Vero E6 cells. Aimed at generating analogues by metabolic engineering, the valinomycin biosynthetic gene cluster has been cloned from Streptomyces tsusimaensis ATCC 15141. Targeted disruption of a nonribosomal peptide synthetase (NRPS) gene abolishes valinomycin production, which confirms its predicted nonribosomal-peptide origin. Sequence analysis of the NRPS system reveals four distinctive modules, two of which contain unusual domain organizations that are presumably involved in the generation of biosynthetic precursors D-alpha-hydroxyisovaleric acid and L-lactic acid. The respective adenylation domains in these two modules contain novel substrate-specificity-conferring codes that might specify for a class of hydroxyl acids for the biosynthesis of the depsipeptide natural products.

Streptomyces genes involved in biosynthesis of the peptide antibiotic valinomycin.

We have identified genes from Streptomyces levoris A-9 involved in the biosynthesis of the peptide antibiotic valinomycin. Two segments of chromosomal DNA were recovered from genomic libraries, constructed by using the low-copy-number plasmid pIJ922, by complementation of valinomycin-deficient (vlm) mutants of S. levoris A-9. One set of plasmids restored valinomycin production to only one mutant, that carrying vlm-1, whereas a second set of plasmids restored productivity to seven vlm mutants, those carrying vlm-2 through vlm-8. Additional complementation studies using subcloned restriction enzyme fragments showed that the vlm-1+ gene was contained within a 2.5-kilobase (kb) DNA region, whereas alleles vlm-2+ through vlm-8+ were contained in a 12-kb region, representing at least three genes. Physical mapping experiments based on the isolation of cosmid clones showed that the two vlm loci were 50 to 70 kb apart. Southern hybridization experiments demonstrated that the vlm-2+ gene cluster was highly conserved among other valinomycin-producing Streptomyces strains, whereas the vlm-1+ gene was ubiquitous among Streptomyces species tested. Increasing the copy number of the vlm-2+ gene cluster in S. levoris A-9 by the introduction of low-copy-number recombinant plasmids resulted in a concomitant increase in the level of valinomycin production.

[Valinomycin biosynthesis and the dynamics of the content of macroergic phosphorus compounds in Streptomyces cyaneofuscatus]. / Biosintez valinomitsina i dinamika soderzhaniia makroérgicheskikh fosfornykh soedinenii u Streptomyces cyaneofuscatus.

The pattern of accumulation and consumption of macroergic phosphoric compounds such as polyphosphates, pyrophosphate and ATP in the mycelium of the valinomycin-producing organism was studied. The content of high polymeric polyphosphates in the high productive strain A of S. cyaneofuscatus was much lower than that in the isogenic low productive strain B, which was indicative of their participation in providing bioenergetics of antibiotic production. Cultivation of the low productive strain B in the presence or absence of the A-factor showed that the mutant reduced its sporulation and provided a 40-fold increase in biosynthesis of valinomycin. However, no difference in consumption of diverse polyphosphate fractions was observed.

[Regulators of differentiation in Streptomyces cyaneofuscatus]. / Reguliatory differentsiatsii Streptomyces cyaneofuscatus.

Streptomyces cyaneofuscatus PRL 1642 producing valinomycin was shown to synthesize bioregulators of differentiation similar to A factor. The regulators stimulate spore formation and streptomycin synthesis in a Streptomyces griseus 1439 mutant deficient in A factor. Some S. cyaneofuscatus mutants respond to A factor addition into the medium by an increase in valinomycin synthesis and a change in morphological properties. The regulators from S. cyaneofuscatus are more effective toward mutants of this species than toward S. griseus mutants.