Enhancement of salinomycin production by ribosome engineering in Streptomyces albus

正Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bacteria are spreading at alarming rates. Natural active products and their derivatives will play     

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization,fast growth,abundant precursors and energy supply,and a pronounced gene expression.Streptomyces albus BK3-25 is a high-yield industrial strain producing type-Ⅰ polyketide sahnomycin,with a unique ability of bean oil utilization.Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein.Firstly,introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and sahnomycin,and subsequent deletion of the sahnomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors,including malonyl-CoA,methylmalonyl-CoA,and ethylmalonyl-CoA.Secondly,the energy and reducing force were measured,and the improved accumulation of ATP and NADPH was observed in the mutant.Furthermore,the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases,and a strong constitutive promoter was identified,providing a useful tool for further engineered gene expression.Finally,the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor,and the heterologous production of actinorhodin was obtained.This work clearly indicated the potential of the high-yield sahnomycin producer as a surrogate host for heterologous production of polyketides,although more genetic manipulation should be conducted to streamline its performance.     

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo. The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.     

Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor

FK506 is a 23-membered polyketide macrolide with immunosuppressant activity produced by Streptomyces species. The production of FK506 in S. clavuligerus CKD1119 (KCTC 10561BP) was improved by enhancing the supply of biosynthetic precursors. This improvement was approximately 2.5-fold (15 mg/l) with the supplementation of 10 mM methyl oleate, which is the probable source of acyl-CoAs, to R2YE medium. When the level of FK506 production reached its maximum, the intracellular concentration of methylmalonyl-CoA in S. clavuligerus CKD1119 supplemented with methyl oleate was 12.5-fold higher than that of the unsupplemented strain, suggesting that an increased methylmalonyl-CoA level caused the high-level production of FK506. The following three pathways for the production of (2S)-methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of FK506 in S. clavuligerus CKD1119: propionyl-CoA carboxylase, methylmalonyl-CoA mutase (MCM), and malonyl/methylmalonyl-CoA ligase. Of the three pathways examined, the MCM pathway supported the highest levels of FK506 production. The expression of MCM in S. clavuligerus CKD1119 led to a threefold and 1.5-fold increase in the methylmalonyl-CoA pool and FK506 production, respectively. Supplementing the culture broth of S. clavuligerus CKD1119 expressing MCM with methyl oleate resulted in an additional twofold increase in the FK506 titer (17.8 mg/l). Overall, these results show that the methylmalonyl-CoA supply is a limiting factor for FK506 biosynthesis and that among the three pathways analyzed, the MCM pathway is the most effective precursor supply pathway supporting the highest titer of FK506 in S. clavuligerus CKD1119.     

Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH , encoding a hybrid polyketide and non-ribosomal pep-tide enzyme. Tandem ketosynthase (KS) domains (KS_10–1 and KS_10–2) were characterized and they show significant homol-ogy with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains totest the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS_10–2 is essential for OZM biosynthesis but KS_10–1 is not indispensable and might serve as a "redundant" domain. These results confirmed the existence of an "extra domain" in complex polyketide synthase.     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

Iteratively improving natamycin production in Streptomyces gilvosporeus by a large operon-reporter based strategy

Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus. In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding. In three successive rounds of mutagenesis and selection, the natamycin titer was increased by 110%, 230%, and 340%, respectively, and the expression of the whole biosynthetic gene cluster was correspondingly increased. An additional copy of the natamycin gene cluster was found in one overproducer. These findings support the large operon reporter-based selection system as a useful tool for the improvement of industrial strains utilized in the production of polyketides and non-ribosomal peptides.     

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS1⁰) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Δ), rather than using the KS1⁰ system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Δ rather than the KS1⁰ system.     

Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from<Emphasis Type="Italic"> Streptomyces coelicolor</Emphasis> A3(2)

Sequence analysis of the metabolically rich 8.7-Mbp genome of the model actinomycete Streptomyces coelicolor A3(2) revealed three genes encoding predicted type III polyketide synthases (PKSs). We report the inactivation, expression, and characterization of the type III PKS homologous SCO1206 gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Incubation of recombinant THNS with malonyl-CoA showed THN production, as demonstrated by UV and HPLC analyses. The K_m value for malonyl-CoA and the k_cat value for THN synthesis were determined spectrophotometrically to be 3.58±0.85 µM and 0.48±0.03 min^–1, respectively. The C-terminal region of S. coelicolor THNS, which is longer than most other bacterial and plant type III PKSs, was shortened by 25 amino acid residues and the resulting mutant was shown to be slightly more active (K_m=1.97±0.19 µM, k_cat=0.75±0.04 min^–1) than the wild-type enzyme.     

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

     

Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks

Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.     

Insertional inactivation of mtrX and mtrY genes from the mithramycin gene cluster affects production and growth of the producer organism Streptomyces argillaceus

Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excision nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.     

Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602

We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602.     

Enhanced production of heterologous macrolide aglycones by fed-batch cultivation of Streptomyces coelicolor

A media development program for the enhanced production of macrolide aglycones by Streptomyces coelicolor is described. Shake flask studies utilizing a yeast extract and a bakers' yeast increased production by 200% and 80%, respectively. However, ammonia generation and high pH were identified as potential problems in these enriched media. Studies in pH-controlled fermentors revealed that production stage pH significantly affects macrolide titers, with low pH (5.5) being more productive than high pH (6.5). Implementation of glucose feeding in shake flask cultures reduced ammonia generation and controlled production stage pH, resulting in significantly enhanced productivities. The combined effects of media supplementation and glucose feeding resulted in a three to five-fold overall improvement in total macrolide aglycone titers, and is the first reported high-level (>1 g/l) production of recombinant polyketides in a heterologous host. Journal of Industrial Microbiology & Biotechnology (2002) 28, 297–301 DOI: 10.1038/sj/jim/7000246 Received 06 August 2001/ Accepted in revised form 26 January 2002     

Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida

Mycinamicin, composed of a branched lactone and two sugars, desosamine and mycinose, at the C-5 and C-21 positions, is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against Gram-positive bacteria. The nucleotide sequence (62 kb) of the mycinamicin biosynthetic gene cluster, in which there were 22 open reading frames (ORFs), was completely determined. All of the products from the 22 ORFs are responsible for the biosynthesis of mycinamicin II and self-protection against the compounds synthesized. Central to the cluster is a polyketide synthase locus (mycA), which encodes a seven-module system comprised of five multifunctional proteins. Immediately downstream of mycA, there is a set of genes for desosamine biosynthesis (mydA-G and mycB). Moreover, mydH, whose product is responsible for the biosynthesis of mycinose, lies between mydA and B. On the other hand, eight ORFs were detected upstream of the mycinamicin PKS gene. The myrB, mycG, and mycF genes had already been characterized by Inouye et al. The other five ORFs (mycCI, mycCII, mydI, mycE, and mycD) lie between mycA1 and mycF, and these five genes and mycF are responsible for the biosynthesis of mycinose. In the PKS gene, four regions of KS and AT domains in modules 1, 4, 5, and 6 indicated that it does not show the high GC content typical for Streptomyces genes, nor the unusual frame plot patterns for Streptomyces genes. Methylmalonyl-CoA was used as substrate in the functional units of those four modules. The relationship between the substrate and the unusual frame plot pattern of the KS and AT domains was observed in the other PKS genes, and it is suggested that the KS-AT original region was horizontally transferred into the PKS genes on the chromosomal DNA of several actinomycetes strains.     

Comparative assessment of the effects of salinomycin and monensin on the biodistribution of lead and some essential metal ions in mice,subjected to subacute lead intoxication

In this study, we present a comparative assessment of the effects of two polyether ionophorous antibiotics (monensin and salinomycin) on the concentrations of lead (Pb), cooper (Cu), zinc (Zn) and iron (Fe) in the kidneys, spleen, liver and brain of Pb-intoxicated animals. Our data demonstrated that the intoxication of ICR male mice with Pb salt resulted in a significant accumulation of Pb in all studied organs of the mice compared to the untreated control animals. The biodistribution of the toxic metal was in the order kidneys > spleen > liver > brain. The treatment of the Pb-intoxicated animals with tetraethylammonium salts of monensic and salinomycinic acids significantly decreased the concentration of the toxic metal ion compared to the toxic control. The effect varied in the interval 38% (for kidneys) to 52% (for brain) compared to the toxic control group (Pb). The tetraethylammonium salt of salinomycinic acid was more effective in reducing the Pb concentration in the brain of the Pb-treated mice compared to monensin. Pb-intoxication did not affect significantly the Zn endogenous concentration compared to the normal values. The treatment of ICR male mice with Pb-salt decreased the Cu concentration in the spleen and increased the Cu concentration in the liver compared to the untreated control animals. The detoxification of the Pb-intoxicated mice with tetraethylammonium salts of salinomycinic and monensic acids restored the Cu concentration in the spleen, but did not affect the Cu levels in the liver. The Pb-intoxication of the ICR mice resulted in a significant decrease of the Fe-concentration in the spleen and liver compared to the untreated control animals. The administration of the tetraethylammonium salts of salinomycinic and monensic acids to the Pb-treated animals restored the levels of Fe in both organs.     

Analysis of NADPH supply during xylitol production by engineered Escherichia coli

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.