Acarbose glycosylation by AcbE for the production of acarstatins with enhanced α-amylase inhibitory activity.

Acarbose is a potent glycosidase inhibitor widely used in the clinical treatment of type 2 diabetes mellitus (T2DM). Various acarbose analogs have been identified while exploring compounds with improved pharmacological properties. In this study, we found that AcbE from Actinoplanes sp. SE50/110 catalyzes the production of acarbose analogs that exhibit significantly improved inhibitory activity towards α-amylase than acarbose. Recombinant AcbE mainly catalyzed the formation of two new compounds, namely acarstatins A and B, using acarbose as substrate. Using high-resolution mass spectrometry, nuclear magnetic resonance, and glycosidase hydrolysis, we elucidated their chemical structures as O-α-d-maltosyl-(1 â†’ 4)-acarbose and O-α-d-maltotriosyl-(1 â†’ 4)-acarbose, respectively. Acarstatins A and B exhibited 1584- and 1478-fold greater inhibitory activity towards human salivary α-amylase than acarbose. Furthermore, both acarstatins A and B exhibited complete resistance to microbiome-derived acarbose kinase 1-mediated phosphorylation and partial resistance to acarbose-preferred glucosidase-mediated hydrolysis. Therefore, acarstatins A and B have great potential as candidate therapeutic agents for T2DM.

Coupled strategy based on regulator manipulation and medium optimization empowers the biosynthetic overproduction of lincomycin.

The biosynthesis of bioactive secondary metabolites, specifically antibiotics, is of great scientific and economic importance. The control of antibiotic production typically involves different processes and molecular mechanism. Despite numerous efforts to improve antibiotic yields, joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite. Lincomycin A (Lin-A), a lincosamide antibiotic, is industrially fermented by Streptomyces lincolnensis. Herein, the leucine-responsive regulatory protein (Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis, whereas indirectly controlled the transcription of SLCG_2919, the first reported repressor in S. lincolnensis. Inactivation of SLCG_4846 in the high-yield S. lincolnensis LA219X (LA219XΔ4846) increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment. Given the effect of the double deletion on cellular primary metabolism of S. lincolnensis, Plackett-Burman design, steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask, and Lin-A yield using optimal seed medium was significantly increased over the control. Above strategies were performed in a 15-L fermenter. The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h, 55.1 % higher than that in LA219X at the parental cultivation (4.23 g/L). This study not only showcases the potential of this strategy to boost lincomycin production, but also could empower the development of high-performance actinomycetes for other antibiotics.

Streptomyces-based whole-cell biosensors for detecting diverse cell envelope-targeting antibiotics.

Cell envelope-targeting antibiotics are potent therapeutic agents against various bacterial infections. The emergence of multiple antibiotic-resistant strains underscores the significance of identifying potent antimicrobials specifically targeting the cell envelope. However, current drug screening approaches are tedious and lack sufficient specificity and sensitivity, warranting the development of more efficient methods. Genetic circuit-based whole-cell biosensors hold great promise for targeted drug discovery from natural products. Here, we performed comparative transcriptomic analysis of Streptomyces coelicolor M1146 exposed to diverse cell envelope-targeting antibiotics, aiming to identify regulatory elements involved in perceiving and responding to these compounds. Differential gene expression analysis revealed significant activation of VanS/R two-component system in response to the glycopeptide class of cell envelope-acting antibiotics. Therefore, we engineered a pair of VanS/R-based biosensors that exhibit functional complementarity and possess exceptional sensitivity and specificity for glycopeptides detection. Additionally, through promoter screening and characterization, we expanded the biosensor's detection range to include various cell envelope-acting antibiotics beyond glycopeptides. Our genetically engineered biosensor exhibits superior performance, including a dynamic range of up to 887-fold for detecting subtle antibiotic concentration changes in a rapid 2-h response time, enabling high-throughput screening of natural product libraries for antimicrobial agents targeting the bacterial cell envelope.

Biosynthesis of ansamitocin P-3 incurs stress on the producing strain Actinosynnema pretiosum at multiple targets.

Microbial bioactive natural products mediate ecologically beneficial functions to the producing strains, and have been widely used in clinic and agriculture with clearly defined targets and underlying mechanisms. However, the physiological effects of their biosynthesis on the producing strains remain largely unknown. The antitumor ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum ATCC 31280, was found to repress the growth of the producing strain at high concentration and target the FtsZ protein involved in cell division. Previous work suggested the presence of additional cryptic targets of AP-3 in ATCC 31280. Herein we use chemoproteomic approach with an AP-3-derived photoaffinity probe to profile the proteome-wide interactions of AP-3. AP-3 exhibits specific bindings to the seemingly unrelated deoxythymidine diphosphate glucose-4,6-dehydratase, aldehyde dehydrogenase, and flavin-dependent thymidylate synthase, which are involved in cell wall assembly, central carbon metabolism and nucleotide biosynthesis, respectively. AP-3 functions as a non-competitive inhibitor of all three above target proteins, generating physiological stress on the producing strain through interfering diverse metabolic pathways. Overexpression of these target proteins increases strain biomass and markedly boosts AP-3 titers. This finding demonstrates that identification and engineering of cryptic targets of bioactive natural products can lead to in-depth understanding of microbial physiology and improved product titers.

Insertion sequence transposition inactivates CRISPR-Cas immunity.

CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.

Isolation, structural characterization and sporicidal properties of Baelezcin A, a novel cyclic lipopeptide from Bacillus velezensis SJ100083 against gray mold.

BACKGROUND: Postharvest gray mold induced by Botrytis cinerea seriously affects cherry quality, resulting in huge economic losses. The aim of this study was to isolate and purify a novel antifungal compound from the endophytic Bacillus velezensis SJ100083 of cherries to prevent postharvest gray mold. RESULTS: In this study, Baelezcin A, extracted and purified from Bacillus velezensis SJ100083, was found effective in suppressing gray mold on cherries. Furthermore, the structure of Baelezcin A was identified as a novel cyclic lipopeptide with molecular formula of C52 H91 N7 O13 through ultra-high-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) and nuclear magnetic resonance (NMR). Baelezcin A treatment at 25 mg L-1 significantly decreased the disease incidence and severity of cherry gray mold, the antifungal mechanism of which was attributed to the accumulation of reactive oxygen species within the spores and the leakage of mycelium cytoplasmal contents, resulting in a low rate of spore germination. Moreover, it was proven to be biologically safe within a certain range by MTT assays. CONCLUSION: Our study demonstrated that Baelezcin A from the culture of Bacillus velezensis SJ100083 may be a promising fruit preservative for controlling postharvest gray mold caused by Botrytis cinerea. © 2023 Society of Chemical Industry.

Multifunctional Protein Hybrid Nanoplatform for Synergetic Photodynamic-Chemotherapy of Malignant Carcinoma by Homologous Targeting Combined with Oxygen Transport.

Photodynamic therapy (PDT) under hypoxic conditions and drug resistance in chemotherapy are perplexing problems in anti-tumor treatment. In addition, central nervous system neoplasm-targeted nanoplatforms are urgently required. To address these issues, a new multi-functional protein hybrid nanoplatform is designed, consisting of transferrin (TFR) as the multicategory solid tumor recognizer and hemoglobin for oxygen supply (ODP-TH). This protein hybrid framework encapsulates the photosensitizer protoporphyrin IX (PpIX) and chemotherapeutic agent doxorubicin (Dox), which are attached by a glutathione-responsive disulfide bond. Mechanistically, ODP-TH crosses the blood-brain barrier (BBB) and specifically aggregated in hypoxic tumors via protein homology recognition. Oxygen and encapsulated drugs ultimately promote a therapeutic effect by down-regulating the abundance of multidrug resistance gene 1 (MDR1) and hypoxia-inducible factor-1-α (HIF-1α). The results reveal that ODP-TH achieves oxygen transport and protein homology recognition in the hypoxic tumor occupation. Indeed, compared with traditional photodynamic chemotherapy, ODP-TH achieves a more efficient tumor-inhibiting effect. This study not only overcomes the hypoxia-related inhibition in combination therapy by targeted oxygen transport but also achieves an effective treatment of multiple tumors, such as breast cancer and glioma, providing a new concept for the construction of a promising multi-functional targeted and intensive anti-tumor nanoplatform.

Crystallographic Deciphering of Spontaneous Self-Assembly of Achiral Calciphores to Chiral Complexes.

Thiapyricins (TPC-A/B, 1 and 2), which are new metallophore scaffolds exhibiting selective divalent cation binding property, were produced in response to metal-deprived conditions by Saccharothrix sp. TRM_47004 isolated from the Lop Nor Salt Lake. TPCs represent a thiazolyl-pyridine skeleton of a calcium-binding natural product, calciphore, owing to the selectivity to calcium ions among diverse metal ions. The thiapyricins exhibited notable co-crystalline characteristics of the apo- and holo-forms with racemic enantiomers comprising a pair of space isomers in a Δ/Λ-form. Therefore, we postulated a mechanism for the four-hierarchical self-assembly of achiral natural products into chiral complexes. Furthermore, their metal-chelating trait aided the adaptation of the host during metal starvation by increasing the production of TPCs. This study presents a structural paradigm of a new calciphore, provides insight into the mechanism of natural product assembly, and highlights the causality between the production of the metallophore and metallic habitats.

Endowing homodimeric carbamoyltransferase GdmN with iterative functions through structural characterization and mechanistic studies.

Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7-O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.

Extraction and characterization of cyclic lipopeptides with antifungal and antioxidant activities from Bacillus amyloliquefaciens.

AIMS: This study aimed to isolate active substances from metabolites of Bacillus amyloliquefaciens SJ100001 and examine their antifungal activity against Fusarium oxysporum (F. oxysporum) SJ300024 screened from the root-soil of cucumber wilt. METHODS AND RESULTS: An active substance, anti-SJ300024, was obtained from the fermentation broth of strain SJ100001 by reversed-phase silica gel and gel chromatography, and further got its chemical structure as cyclic lipopeptide Epichlicin through nuclear magnetic resonance (NMR) and mass spectrometry (MS). In vitro experiments showed that Epichlicin had a better inhibitory rate (67.46%) against the strain SJ300024 than the commercially available fungicide hymexazol (45.10%) at the same concentration. The MTT assays proved that Epichlicin was non-cytotoxic, besides it also had good free radical scavenging ability and total reducing ability. CONCLUSIONS: Epichlicin isolated from strain SJ100001 can effectively control F. oxysporum SJ300024 screened from the root-soil of cucumber wilt. SIGNIFICANCE AND IMPACT OF THE STUDY: Epichlicin may be used as an environmentally friendly and efficient biocontrol agent for controlling Fusarium wilt of cucumber and reducing crop losses. More importantly, the non-cytotoxicity of Epichlicin can avoid harm to consumers. Additionally, Epichlicin has broad application prospects in medicine due to its antioxidant properties.

Comparative Transcriptome-Based Mining of Genes Involved in the Export of Polyether Antibiotics for Titer Improvement.

The anti-coccidiosis agent salinomycin is a polyether antibiotic produced by Streptomyces albus BK3-25 with a remarkable titer of 18 g/L at flask scale, suggesting a highly efficient export system. It is worth identifying the involved exporter genes for further titer improvement. In this study, a titer gradient was achieved by varying soybean oil concentrations in a fermentation medium, and the corresponding transcriptomes were studied. Comparative transcriptomic analysis identified eight putative transporter genes, whose transcription increased when the oil content was increased and ranked top among up-regulated genes at higher oil concentrations. All eight genes were proved to be positively involved in salinomycin export through gene deletion and trans-complementation in the mutants, and they showed constitutive expression in the early growth stage, whose overexpression in BK3-25 led to a 7.20-69.75% titer increase in salinomycin. Furthermore, the heterologous expression of SLNHY_0929 or SLNHY_1893 rendered the host Streptomyces lividans with improved resistance to salinomycin. Interestingly, SLNHY_0929 was found to be a polyether-specific transporter because the titers of monensin, lasalocid, and nigericin were also increased by 124.6%, 60.4%, and 77.5%, respectively, through its overexpression in the corresponding producing strains. In conclusion, a transcriptome-based strategy was developed to mine genes involved in salinomycin export, which may pave the way for further salinomycin titer improvement and the identification of transporter genes involved in the biosynthesis of other antibiotics.

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin.

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein-protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein-protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein-protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

Antibacterial Prenylated p-Hydroxybenzoic Acid Derivatives from Oberonia myosurus and Identification of Putative Prenyltransferases.

Twelve hitherto unknown tandem prenylated p-hydroxybenzoic acid derivatives, namely, oberoniamyosurusins A-L, together with five known derivatives, were isolated from an EtOH extract of the whole parts of the plant Oberonia myosurus. Compounds 10, 13, and 17 exhibited moderate inhibitory activity against Staphylococcus aureus subsp. aureus ATCC29213 with MIC50 values ranging from 7.6 to 23 µg/mL. To determine the biosynthetic pathway of this class of tandem prenyl-substituted compounds, the full-length transcriptome of O. myosurus was sequenced, yielding 19.09 Gb of clean data and 10 949 nonredundant sequences. Two isoforms of p-hydroxybenzoic acid prenyltransferases were annotated and functionally characterized as the enzymes that might be involved in the biosynthesis of nervogenic acid (13) in Pichia pastoris.

p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation.

Actinosynnema species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two Actinosynnema pretiosum subsp. auranticum strains DSM 44131T (hereafter abbreviated as strain DSM 44131T) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131T. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, p-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of dinV, dinE and dinF in Escherichia coli. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131T were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with p-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.

Nonomuraea nitratireducens sp. nov., a new actinobacterium isolated from Suaeda australis Moq. rhizosphere.

A novel actinomycete, designated WYY166T, was isolated from the rhizosphere of Suaeda australis Moq. collected in Dongfang, PR China. The taxonomic position of this strain was investigated using a polyphasic approach. Phylogenetic analysis based on its 16S rRNA gene referred strain WYY166T to the genus Nonomuraea, and it was most closely related to the type strains Nonomuraea candida HMC10T, Nonomuraea turkmeniaca DSM 43926T, Nonomuraea maritima NBRC 106687T and Nonomuraea polychroma DSM 43925T (98.35, 97.60, 97.36 and 97.30% sequence similarity, respectively). Genome sequencing revealed a genome size of 11.27 Mbp and a G+C content of 71.10 mol%. The genome average nucleotide identity (ANI) values and the digital DNA - DNA hybridization (dDDH) values between strain WYY166T and the other species of the genus were found to be low (ANI 81.63~85.23 %, dDDH 23.6~31.6 %), suggesting that it represented a new species. The physiological evaluation showed that it had remarkable nitrate reduction activity. The whole-cell hydrolysates contained meso-diaminopimelic acid and madurose. The N-acyl type of muramic acid was acetyl. The major menaquinones were MK-9 (H4) (86.9 %) and MK-9 (H2) (13.1 %). The predominant fatty acids were iso-C16 : 0 (53.2 %), 10-methyl C17 : 0 (10.7 %), C17 : 1 ω6c (8.3 %) and iso-C16 : 1 h (7.3 %). These physiological, biochemical and chemotaxonomic data suggested that strain WYY166T should be classified as representing a novel species of the genus Nonomuraea, for which the name Nonomuraea nitratireducens sp. nov. is proposed. The type strain is WYY166T (=MCCC 1K03779T=KCTC 49343T).

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes.

In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.

The Antitumor Agent Ansamitocin P-3 Binds to Cell Division Protein FtsZ in Actinosynnema pretiosum.

Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards ß-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of ß-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM_5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.

A severe leakage of intermediates to shunt products in acarbose biosynthesis.

The α-glucosidase inhibitor acarbose, produced by Actinoplanes sp. SE50/110, is a well-known drug for the treatment of type 2 diabetes mellitus. However, the largely unexplored biosynthetic mechanism of this compound has impeded further titer improvement. Herein, we uncover that 1-epi-valienol and valienol, accumulated in the fermentation broth at a strikingly high molar ratio to acarbose, are shunt products that are not directly involved in acarbose biosynthesis. Additionally, we find that inefficient biosynthesis of the amino-deoxyhexose moiety plays a role in the formation of these shunt products. Therefore, strategies to minimize the flux to the shunt products and to maximize the supply of the amino-deoxyhexose moiety are implemented, which increase the acarbose titer by 1.2-fold to 7.4 g L-1. This work provides insights into the biosynthesis of the C7-cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.

Generation of tetramycin B derivative with improved pharmacological property based on pathway engineering.

Polyene antibiotics, including amphotericin, nystatin, pimaricin, and tetramycin, are important antifungal agents. Increasing the production of polyenes and generation of their improved analogues based on the biosynthetic pathway engineering has aroused wide concern in application researches. Herein, tetramycin and nystatin, both of which share most of acyl-CoA precursors, are produced by Streptomyces hygrospinosus var. beijingensis CGMCC 4.1123. Thus, the intracellular malonyl-CoA is found to be insufficient for PKSs (polyketide synthases) extension of tetramycin by quantitative analysis in this wild-type strain. To circumvent this problem and increase tetramycin titer, the acyl-CoA competing biosynthetic gene cluster (BGC) of nystatin was disrupted, and the biosynthetic genes of malonyl-CoA from S. coelicolor M145 were integrated and overexpressed in nys-disruption mutant strain (SY02). Moreover, in order to specifically accumulate tetramycin B from A, two copies of tetrK and a copy of tetrF were introduced, resulting in elevating tetramycin B fermentration titer by 122% to 865 ± 8 mg/L than the wild type. In this optimized strain, a new tetramycin derivative, 12-decarboxy-12-methyl tetramycin B, was generated with a titer of 371 ± 26 mg/L through inactivation of a P450 monooxygenase gene tetrG. Compared with tetramycin B, the new compound exhibited higher antifungal activity against Saccharomyces cerevisiae and Rhodotorula glutinis, but lower hemolytic toxicity to erythrocyte. This research provided a good example of employing biosynthetic engineering strategies for fermentation titer improvement of polyene and development of the derivatives for medicinal applications.