Tetracycline Biosynthesis Research Articles

Managing tobacco black shank disease using biochar: direct toxicity and indirect ecological mechanisms.

Black shank disease in tobacco, caused by Phytophthora nicotianae, can lead to yield losses of 30%-50% upon outbreak. Recently, biochar derived from agricultural waste has shown significant potential in controlling soil-borne diseases, though its mechanisms remain unclear. Over a 3-year observation period, we found that the incidence of black shank was significantly lower in plots amended with biochar compared with normal cultivation plots. To investigate the underlying mechanisms, we studied both the direct and indirect effects of biochar on black shank. Direct antifungal assays indicated that biochar reduced the total number of sporangia by 53.91%. Further pot experiments revealed a 62.34% reduction in the P. nicotianae population in the soil following biochar application. Additionally, biochar application led to notable changes in soil physicochemical properties and microbial community composition. Microbial species analysis showed that biochar promoted the aggregation of beneficial microbes such as Sphingomonas, Flavisolibacter, and Mucoromycota. Functional predictions using the PICRUSt 2 software revealed that biochar enhances bacterial functions related to antimicrobial substance synthesis (Tetracycline biosynthesis), detoxification metabolism (D-arginine and D-ornithine metabolism, arginine and proline metabolism), and lipid and fatty acid metabolism (Lipopolysaccharide biosynthesis, fatty acid biosynthesis), while fungal functions showed no significant changes. This suggests that rhizosphere bacteria play a more prominent role in the suppression of black shank by biochar, a finding supported by partial least squares path modeling analysis. Therefore, we hypothesize that biochar not only directly inhibits P. nicotianae growth but also regulates the composition of the rhizosphere microbial community, inducing the production of antimicrobial substances by rhizosphere bacteria, effectively preventing P. nicotianae invasion.IMPORTANCEBlack shank, a global soil-borne fungal disease in tobacco, currently lacks effective control methods. Notably, biochar derived from agricultural waste has shown significant potential in controlling soil-borne diseases. Over a 3-year observation period, we found that plots amended with biochar had a significantly lower incidence of black shank compared with normal cultivation plots. However, the mechanisms of disease suppression remained unclear. Through in vitro antifungal assays and pot experiments, we discovered that tobacco-derived biochar can directly inhibit the growth of the pathogen. Additionally, biochar regulates the composition of the rhizosphere microbial community, inducing rhizosphere bacteria to produce antimicrobial substances, effectively preventing pathogen invasion. This discovery reveals both the direct and indirect mechanisms by which biochar suppresses black shank in tobacco. It provides a scientific basis for developing green control technologies for black shank and offers theoretical support for the application of biochar in managing soil-borne diseases in tobacco cultivation areas.

Metagenomics reveals microbial communities and functional differences during chili pepper (Capsicum frutescens L.) natural fermentation: Effects of brines and containers

AbstractThe study aimed to investigate the effect of fermentation brines and containers on the composition and functional dissimilarity of microbiota in fermented chili peppers: pool with fresh brine (PFB), pool with aged brine (PAB), jar with FB, and jar with AB. The results showed that brines had a greater impact on microbial diversity and fungal community structure than containers, whereas containers had a greater impact on bacterial community structure than brines. At the end of chili pepper fermentation, the dominant bacteria were unclassified_g__Pseudomonas, Lactobacillus_alimentarius, and Halococcus_thailandensis, whereas the dominant fungi were Pichia_membranifaciens and Wallemia_ichthyophaga. Based on the metagenomics functional analysis, the dominant bacteria and fungi were highly related to amino acid metabolism, glycerophospholipid metabolism, and most enzymes that promote carbohydrate substrate modulation. The fermented brines had a larger impact than containers on microbial function. Specifically, the use of FB was found to be beneficial for the biosynthesis of tetracycline, carotenoid, and isoflavonoid. On the other hand, AB was found to be beneficial for the biosynthesis of unsaturated fatty acids, amino acids, and various types of N‐glycan.

Effects of Rhizosphere Microbial Communities on Cucumber Fusarium wilt Disease Suppression.

Cucumber Fusarium wilt is a worldwide soil-borne disease that seriously restricts the yield and quality of cucumber. The rhizosphere soil microbiome, as the first line of defense against pathogens invading plant roots, plays a key role in rhizosphere immune formation and function. The purpose of this study was to reveal the key microecological factors and dominant microbial flora affecting cucumber resistance and susceptibility to Fusarium wilt by analyzing the physical and chemical properties and microbial flora of rhizosphere soil with different degrees of susceptibility and resistance to cucumber Fusarium wilt, thereby laying a foundation to establish cucumber resistance to the Fusarium wilt rhizosphere core microbiome. Firstly, Illumina Miseq sequencing technology was used to evaluate the physical and chemical properties and microbial groups of cucumber rhizosphere soil at different health levels, and the key environmental factors and microbial factors related to cucumber Fusarium wilt were screened out. Subsequently, PICRUSt2 and FUNGuild were used to predict the functions of rhizosphere bacteria and fungi. Combined with functional analysis, the possible interactions among soil physical and chemical properties, cucumber rhizosphere microorganisms, and Fusarium wilt were summarized. The results showed that the available potassium content in the rhizosphere soil of healthy cucumber decreased by 10.37% and 0.56%, respectively, compared with the rhizosphere soil of severely susceptible cucumber and mildly susceptible cucumber. Exchangeable calcium content increased by 25.55% and 5.39%; the α diversity Chao1 index of bacteria and fungi in the rhizosphere soil of healthy cucumber was significantly lower than that in the rhizosphere soil of seriously infected cucumber, and the MBC content of its physical and chemical properties was also significantly lower than that in the rhizosphere soil of seriously infected cucumber. There was no significant difference in the Shannon and Simpson diversity indexes between healthy cucumber rhizosphere soil and seriously infected cucumber rhizosphere soil. The results of the β diversity analysis showed that the bacterial and fungal community structure of healthy cucumber rhizosphere soil was significantly different from that of severely and mildly infected cucumber rhizosphere soil. At the genus level, through statistical analysis, LEfSe analysis, and RDA analysis, the key bacterial and fungal genera with potential biomarker values were screened out as SHA_26, Subgroup_22, MND1, Aeromicrobium, TM7a, Pseudorhodoplanes, Kocuria, Chaetomium, Fusarium, Olpidium, and Scopulariopsis, respectively. The bacteria SHA_26, Subgroup_22, and MND1 related to cucumber Fusarium wilt inhibition belong to Chloroflexi, Acidobacteriota, and Proteobacteria, respectively. Chaetomiacea belongs to Sordariomycates. The results of functional prediction showed that changes to the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway in the bacterial microbiota were concentrated in tetracycline biosynthesis, selenocompound metabolism, lipopolysaccharide biosynthesis, etc., which were mainly involved in the metabolism of terpenoids and polyketides, energy metabolism, metabolism of other amino acids, glycan biosynthesis and metabolism, lipid metabolism, cell growth and death, transcription, metabolism of cofactors and vitamins, and biosynthesis of other secondary metabolites. The difference in fungi was mainly dung saprotroph-ectomycorrhizal-soil saprotroph-wood saprotroph. Through the correlation analysis and functional predictions of the key environmental factors, microbial flora, and cucumber health index in cucumber rhizosphere soil, we determined that the inhibition of cucumber Fusarium wilt was a synergistic effect of environmental factors and microbial flora, and a model diagram was drawn to briefly explain its mechanism. This work will provide a basis for the biological control of cucumber Fusarium wilt in the future.

Dietary supplementation with Chinese herb ultrafine powder improves intestinal morphology and physical barrier function by altering jejunal microbiota in laying hens.

Chinese medicinal herbs play important roles in anti-inflammatory, antioxidant, and antibacterial activities. However, the effects of Chinese herb ultrafine powder (CHUP) on laying hens still need to be elucidated. Therefore, this study aimed to evaluate the effects of dietary CHUP supplementation on jejunal morphology, physical barrier function, and microbiota in laying hens. A total of 576 Xinyang black-feather laying hens (300 days old) were randomly assigned into eight groups, with eight replicates per group and nine hens per replicate. The hens were fed a basal diet (control group) and a basal diet supplemented with 0.5% Leonuri herba (LH group), 0.25% Ligustri lucidi fructus (LF group), 0.25% Taraxaci herba (TH group), 0.5% LH + 0.25% LF (LH-LF group), 0.5% LH + 0.25% TH (LH-TH group), 0.25% LF + 0.25% TH (LF-TH group), and 0.5% LH + 0.25% LF + 0.25% TH (LH-LF-TH group), respectively, for 120 days. The results showed that dietary LH-LF and LH-LF-TH supplementation increased (p < 0.05) the jejunal villus height to crypt depth ratio of laying hens. Dietary LF-TH supplementation up-regulated jejunal claudin-5 expression, while LH supplementation up-regulated jejunal claudin-1 expression and increased the jejunal abundances of potentially beneficial bacteria related to short-chain fatty acids and bacteriocins production, such as Blautia, Carnobacterium, Clostridiales, and Erysipelotrichales (p < 0.05). In addition, dietary LH supplementation enriched (p < 0.05) the tetracycline biosynthesis, butirosin/neomycin biosynthesis, and D-arginine/D-ornithine metabolism, whereas steroid biosynthesis and limonene/pinene degradation were enriched (p < 0.05) in the LH-LF and LH-LF-TH groups. Moreover, Spearman's correlation analysis revealed the potential correlation between the abundance of the jejunal microbiota and jejunal morphology and the physical barrier function of laying hens. Collectively, these findings suggest that dietary CHUP supplementation could enhance the beneficial bacteria abundance, physical barrier function, and metabolic function associated with short-chain fatty acids and bacteriocins production. Moreover, combined supplementation of dietary CHUP showed better effects than the sole CHUP supplementation.

The functional microbiota of on- and off-year moso bamboo (Phyllostachys edulis) influences the development of the bamboo pest Pantana phyllostachysae

BackgroundDevelopment of Pantana phyllostachysae, a moso bamboo pest, is affected by its diet. Understanding the mechanism underlying the different insect-resistant capacities of on- and off-year moso bamboo fed by P. phyllostachysae is crucial for managing pest outbreaks. As microbes were proven to influence plant immunity, we compared gut microbial communities of P. phyllostachysae with different diets by metabarcoding sequencing. By using sterilization assay, microbes were removed from leaf surfaces, and thus we confirmed that microbes inhabiting moso bamboo leaves impact the weight of P. phyllostachysae larva. Furthermore, the gut microbial communities of P. phyllostachysae fed on on- and off-year bamboo leaves were compared, to identify the functional microbial communities that impact the interaction between bamboo leaves and P. phyllostachysae.ResultsWe found that species from orders Lactobacillales and Rickettsiales are most effective within functional microbiota. Functional prediction revealed that gut microbes of larva fed on on-year leaves were related to naphthalene degradation, while those fed on off-year leaves were related to biosynthesis of ansamycins, polyketide sugar unit biosynthesis, metabolism of xenobiotics, and tetracycline biosynthesis. Most functional microbes are beneficial to the development of larva that feed on on-year bamboo leaves, but damage the balance of intestinal microenvironment and immune systems of those larva that feed on off-year leaves.ConclusionsThis work developed an efficient strategy for microbiome research of Lepidopteran insects and provided insights into microbiota related to the interaction between host plants and P. phyllostachysae. We provided microbial candidates for the ecological control of P. phyllostachysae according to the function of effective microbiota.

Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

Abstract 1780: Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer

Abstract The gut microbiome is now known to influence host immune function, malignancies, and response to therapy. We previously showed that sequencing PD-L1/JAK1/2 blockade prior to androgen deprivation therapy (ADT) could promote immune activity to potentiate antitumor responses in mouse Pten-deficient prostate cancer. Here, we investigate the associations between the treatment pairings of PD-L1 antibody (aPD-L1, clone D265A, mouse/IgG1 kappa) blockade and the JAK1/2 inhibitor AZD1480 with ADT in mouse Pten-deficient prostate cancer and fecal gut microbiomes. We used 16S rRNA amplicon sequencing to survey fecal samples of tumor bearing conditional Pten-knockout (KO) mice treated for four weeks with aPD-L1 and AZD14870 alone and as combination therapy in intact mice, and as concurrent (Conc) therapy with ADT (via surgical castration), or sequenced as adjuvant (Adj) or neoadjuvant (NeoAdj) therapy. In this model, PD-L1 blockade was ineffective as monotherapy in all treatment settings and as combination therapy in intact and Conc combination settings and hyperprogression was observed in some mice treated with aPD-L1 monotherapy in intact and Adj settings. In the NeoAdj setting, aPDL1/AZD1480 therapy demonstrated superiority over monotherapy. Significant differences in microbial composition were observed between groups (P&amp;lt;0.001), drugs (P&amp;lt;0.001), castration status (P&amp;lt;0.001) and treatment responses (P&amp;lt;0.001). AZD1480 as monotherapy or in combination with aPD-L1 was associated with the greatest changes in microbial composition followed by surgical castration. In the neoadjuvant setting Allobaculum, Lactobacillus, Sutturella, Turcibacter and Prevotella were associated with responders whereas Hellicobacter, Oscillospora, and Ruminococcus gnavus were associated with hyperprogression. Functionally, enrichments in fatty acid biosynthesis/metabolism, tetracycline biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and glycolysis/gluconeogenesis were revealed in responders whereas lipopolysaccharide biosynthesis, glycosaminoglycan degradation, fructose and mannose metabolism, histidine metabolism, glyoxylate and dicarboxylate metabolism, and the citrate cycle (TCA) were enriched in low responders. This study provides insight into the complex interactions between gut microbiota and cancer-burdened hosts, and reveals associations between gut microbial composition and treatment responses to androgen deprivation, immune modulation via JAK1/2 inhibition and PD-L1 blockade in a mouse model of Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1780.

A Yeast Three Hybrid Assay for Metabolic Engineering of Tetracycline Derivatives.

Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Toward this end, we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Various tetracycline derivatives can be detected using our assay, whose versatility enables its use both as a screen and a selection to match the needs and instrumentation of a wide range of end users. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and nonproducer strains of the natural product tetracycline TAN-1612. The Y3H assay is superior to state-of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives. Finally, our establishment of the Y3H assay for detecting the biosynthesis of a tetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules.

Cofactor Tail Length Modulates Catalysis of Bacterial F420-Dependent Oxidoreductases

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

Biosynthesis of Oxytetracycline by Streptomyces rimosus: Past, Present and Future Directions in the Development of Tetracycline Antibiotics.

Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of 'broad-spectrum antibiotics' and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickettsiae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third--generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions.

5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.

An ATP-dependent ligase catalyzes the fourth ring cyclization in tetracycline biosynthesis

Cyclization steps in biosynthesis of aromatic polyketides are typically directed by specific enzymes known as cyclases. The fourth ring cyclization step that forms the fully aromatic pretetramid during tetracycline biosynthesis has not been resolved. Herein we report in vitro and in vivo studies on the fourth ring cyclization step in biosynthesis of SF2575 and related tetracyclic natural products. We demonstrate that an enzyme belonging to the ATP-dependent acyl-CoA ligase family catalyzes the C1–C18 Claisen condensation step that forms the A ring and yields the linear, tetracyclic aromatic compound that is the precursor to tetracyclic natural products. The enzyme, SsfL2, is well-conserved in all tetracycline biosynthetic gene clusters discovered to date. It is proposed that SsfL2 directly adenylates the tricyclic carboxylic acid to facilitate the final carbon-carbon bond formation, and coenzyme A is not required.

희소방선균의 seaR 단백질 발현을 통한 기능 분석

Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, -butyrolactone autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a -derived integration vector, pSET152, which contained int, oriT, attP and (erythromycin promotor). Therefore, the pEV615 was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or resistance genes for tetracycline are repressed or not.

Completing our understanding of tetracycline biosynthesis: the enzymatic basis of the F420‐dependent final reduction step.

Tetracyclines are important broad‐spectrum antibiotics. While the discovery of tetracyclines date back to the 1940s, our understanding of the biosynthesis of tetracyclines has only been investigated in recent years. One unknown step in the pathway is the conversion of 5a, 11a‐dehydrotetracycline to either oxytetracycline in Streptomyces rimosus or tetracycline in Streptomyces aureofaciens. The reduction of the 5a–11a double bond is likely catalyzed by a F420‐dependent enzymes, however, the enzymatic basis for this reaction remains unresolved. Using a heterologous expression platform as a starting point, we discovered a small, previously unknown enzyme in both the oxy and ctc pathways that is responsible for this reduction. In vitro reconstitution using this small enzymes, in conjunction with the anhydrotetracycline oxygenase, showed that anhydrotetracycline can be converted to the final products. We also provide biochemical and structural explanation for the differential formation of oxytetracycline and tetracycline from the respective pathways. The result of this work therefore completes our understanding of the tetracycline pathways and pave the way for engineered biosynthesis of analogs.

Identification of OxyE as an Ancillary Oxygenase during Tetracycline Biosynthesis

The double hydroxylation of 6-pretetramid to 4-keto-anhydrotetracycline is a key tailoring reaction during the biosynthesis of the broad-spectrum antibiotic tetracyclines. It has been shown previously by heterologous reconstitution that OxyL is a dioxygenase and is the only enzyme required to catalyze the insertion of oxygen atoms at the C-12a and C-4 positions. We report here that OxyE, a flavin adenine dinucleotide (FAD)-dependent hydroxylase homologue, is an ancillary mono-oxygenase for OxyL during oxytetracycline biosynthesis in Streptomyces rimosus. By using both gene disruption and heterologous reconstitution approaches, we demonstrated that OxyE plays a nonessential, but important role in oxytetracycline biosynthesis by serving as a more efficient C-4 hydroxylase. In addition, we demonstrated that partially oxidized biosynthetic intermediates can undergo various glycosylation modifications in S. rimosus. Our results indicate that the synergistic actions of OxyE and OxyL in the double hydroxylation step prevent accumulation of shunt products during oxytetracycline biosynthesis in S. rimosus.

Decoding and engineering tetracycline biosynthesis

Tetracyclines have been important agents in combating infectious disease since their discovery in the mid-20th century. Following widespread use, tetracycline resistance mechanisms emerged and continue to create a need for new derivatives that are active against resistant bacterial strains. Semisynthesis has led to second and third generation tetracycline derivatives with enhanced antibiotic activity and pharmacological properties. Recent advancement in understanding of the tetracycline biosynthetic pathway may open the door to broaden the range of tetracycline derivatives and afford analogs that are difficult to access by synthetic chemistry.

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.

Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens.

Streptomyces aureofaciens ATCC 10762 grown in rotary-shaken submerged cultures produced substantial amounts of tetracycline only when the defined medium was deprived of iron. The biosynthesis of tetracycline was inhibited either by free iron at concentrations above 1-2 mumol l-1, or by chelated iron provided by the siderophores of this bacterial strain. Late static iron-containing cultures allowed cell differentiation and sporulation and led to tetracyclines synthesis. A nitrosoguanidine-induced mutant able to synthesize tetracycline in the presence of iron in shaken submerged cultures was isolated and compared to the wild-type strain. However, no constitutive siderophore-mediated iron transport occurred in the mutant. These results suggest the involvement of a putative iron-controlled repressor in the biosynthesis of these secondary metabolites during vegetative growth and primary metabolism of the bacterium.

Alizarin glucosyl transferase activity inStreptomyces aureofaciens mutants

Alizarin glucosyl transferase activity was found in five mutant strains of Streptomyces aureofaciens. The activity bears no direct relationship to the final products of tetracycline biosynthesis.

A direct-injection reversed-phase liquid chromatographic micromethod for studying the kinetics of terminal reactions of tetracycline biosynthesis

A new micromethod for measuring enzyme-catalyzed reactions was developed. The method involves a number of consecutive direct injections of aliquots of the reaction mixture onto a microbore column and permits the determination of the time dependence of the decrease of substrate or increase of product concentrations. The reactions proceed in a microvial placed in the autosampler, and as the starting volume can be as low as 10 μl, the requirement for the amount of enzyme is very low. The autosampler backed by the liquid chromatographic software allows automation of the analyses including data processing and easy quantitation of the enzymatic reaction(s). The method was applied to a system of two consecutive terminal reactions of tetracycline biosynthesis in Streptomyces aureofaciens, catalyzed by anhydrotetracycline oxygenase and tetracycline dehydrogenase. The usage of a diode-array detector facilitated the quantification of the reactions as the product and substrate could be monitored at their optimal wavelengths.