- He received his B. Sc. and M. Sc. degree at Northwest Aedit
Angucyclines and angucyclinones are aromatic polyketides with intriguing structures and therapeutic value. Genome mining of the rare marine actinomycete Saccharothrix sp. D09 led to the identification of a type II polyketide synthase... more
Angucyclines and angucyclinones are aromatic polyketides with intriguing structures and therapeutic value. Genome mining of the rare marine actinomycete Saccharothrix sp. D09 led to the identification of a type II polyketide synthase biosynthetic gene cluster, sxn, which encodes several distinct subclasses of oxidoreductases, implying that this strain has the potential to produce novel polycyclic aromatic polyketides with unusual redox modifications. The "one strain-many compounds" (OSMAC) strategy and comparative metabolite analysis facilitated the discovery of 20 angucycline derivatives from the D09 strain, including six new highly oxygenated saccharothrixins D-I (1-6), four new glycosylated saccharothrixins J-M (7-10), and 10 known analogues (11-20). Their structures were elucidated based on detailed HRESIMS, NMR spectroscopic, and X-ray crystallographic analysis. With the help of gene disruption and heterologous expression, we proposed their plausible biosynthetic pathways. In addition, compounds 3, 4, and 8 showed antibacterial activity against Helicobacter pylori with MIC values ranging from 16 to 32 μg/mL. Compound 3 also revealed anti-inflammatory activity by inhibiting the production of NO with an IC50 value of 28 μM.
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Siderophores are secreted by microorganisms to survive in iron-depleted conditions, and they also possess tremendous therapeutic potential. Genomic-inspired isolation facilitated the identification of eight amphiphilic siderophores,... more
Siderophores are secreted by microorganisms to survive in iron-depleted conditions, and they also possess tremendous therapeutic potential. Genomic-inspired isolation facilitated the identification of eight amphiphilic siderophores, saccharochelins A-H (1-8), from a rare marine-derived Saccharothrix species. Saccharochelins feature a series of fatty acyl groups appended to the same tetrapeptide skeleton. With the help of gene disruption and heterologous expression, we identified the saccharochelin biosynthetic pathway. The diversity of saccharochelins originates from the flexible specificity of the starter condensation (CS) domain at the beginning of the nonribosomal peptide synthetase (NRPS) toward various fatty acyl substrates. Saccharochelins showed cytotoxicity against several human tumor cell lines, with IC50 values ranging from 2.3 to 17 μM. Additionally, the fatty acid side chains of the saccharochelins remarkably affected the cytotoxicity, suggesting changing the N-terminal acyl groups of lipopeptides may be a promising approach to produce more potent derivatives.
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Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain... more
Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future.
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The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery... more
The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery of new-to-nature compounds. The two juxtaposed conventional model microorganisms, Escherichia coli, Saccharomyces cerevisiae, have been harnessed as microbial chassis to produce a bounty of secondary metabolites with the help of certain host engineering. In last decade, engineering non-model microbes to efficiently biosynthesize secondary metabolites has received increasing attention due to their peculiar advantages in metabolic networks and/or biosynthesis. The state-of-the-art synthetic biology tools lead the way in operating genetic manipulation in non-model microorganisms for phenotypic optimization or yields improvement of desired secondary metabolites. In this review, we firstly discuss the pros and cons of several model and non-model microbial chassis, as well as the importance of developing broader non-model microorganisms as alternative programmable heterologous hosts to satisfy the desperate needs of biosynthesis study and industrial production. Then we highlight the lately advances in the synthetic biology tools and engineering strategies for optimization of non-model microbial chassis, in particular, the successful applications for efficient heterologous production of multifarious complex secondary metabolites, e.g., polyketides, nonribosomal peptides, as well as ribosomally synthesized and post-translationally modified peptides. Lastly, emphasis is on the perspectives of chassis cells development to access the ideal cell factory in the artificial intelligence-driven genome era.
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Additional file 1: Figure S1. Alignment of the tertiary structures of P450cam and EpoK. Figure S2. Visual representation of the location of the six selected ferredoxin genes and two selected ferredoxin reductase genes. Figure S3. Multiple... more
Additional file 1: Figure S1. Alignment of the tertiary structures of P450cam and EpoK. Figure S2. Visual representation of the location of the six selected ferredoxin genes and two selected ferredoxin reductase genes. Figure S3. Multiple sequence alignment of EpoK from three sources. Figure S4. Results of colony PCR to verify the transformation of plasmids pEOK102 to pEOK116 into H7029-1. Table S1. Yields and component proportion of epothilones in mutants of strain H7029-1. Figure S5. Comparison of the protein sequence and structural alignment between Fdx_0135 and Fdx_A6445. Figure S6. HPLC results of epothilone C crude extracts. Figure S7. Results of colony PCR to verify the transformation of the corresponding plasmids into the wild-type strain DSM 7029. Figure S8. Verification of fdx_0135 knock-out by two-step homologous recombination in strain H7029-1. Figure S9. Comparison of the loss rate of plasmid pEOK114 (oriV-trfA-Aprar-Pkan-epoK-Pkan-fdx_0135-Pkan-fdr_0130) in strains JH0...
Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories.... more
Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional ...
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A cryptic trans-acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide... more
A cryptic trans-acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide shuangdaolides A-D (1-4) and dumulmycin (5). Heterologous expression and gene inactivation experiments allowed the identification of two biosynthetic intermediates, 6 and 7, suggesting an unusual multidomain SDR oxidoreductase SdlR in charge of the formation of a rare 2-hydroxycyclopentenone moiety in this class of compounds.
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Genome mining of Fulvivirga sp. W222 revealed a desferrioxamine-like biosynthetic gene cluster containing an unknown gene fulF that is conserved in many Bacteroidetes species. A series of primary amine-acylated desferrioxamine G1... more
Genome mining of Fulvivirga sp. W222 revealed a desferrioxamine-like biosynthetic gene cluster containing an unknown gene fulF that is conserved in many Bacteroidetes species. A series of primary amine-acylated desferrioxamine G1 analogues, fulvivirgamides, were identified, and fulvivirgamides A2, B2, B3, and B4 (1-4) were purified and characterized. The function of FulF, which is a novel acyltransferase for the acylation of the primary amine of Desferrioxamine G1, was verified by heterologous expression and feeding experiments.
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The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery... more
The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery of new-to-nature compounds. The two juxtaposed conventional model microorganisms, Escherichia coli, Saccharomyces cerevisiae, have been harnessed as microbial chassis to produce a bounty of secondary metabolites with the help of certain host engineering. In last decade, engineering non-model microbes to efficiently biosynthesize secondary metabolites has received increasing attention due to their peculiar advantages in metabolic networks and/or biosynthesis. The state-of-the-art synthetic biology tools lead the way in operating genetic manipulation in non-model microorganisms for phenotypic optimization or yields improvement of desired secondary metabolites. In this review, we firstly discuss the pros and cons of several model and non-model microbial chassis, as well as the importance of developing broader non-model microorganisms as alternative programmable heterologous hosts to satisfy the desperate needs of biosynthesis study and industrial production. Then we highlight the lately advances in the synthetic biology tools and engineering strategies for optimization of non-model microbial chassis, in particular, the successful applications for efficient heterologous production of multifarious complex secondary metabolites, e.g., polyketides, nonribosomal peptides, as well as ribosomally synthesized and post-translationally modified peptides. Lastly, emphasis is on the perspectives of chassis cells development to access the ideal cell factory in the artificial intelligence-driven genome era.
Research Interests:
Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories.... more
Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional mutagenesis at multiple sites on kilobase scales in diverse bacteria without the generation of doublestrand breaks or disturbance of the mismatch repair system. dReaMGE can achieve combinatorial genome engineering works, for example, alterations to multiple biosynthetic pathways, multiple promoter or gene insertions, variations of transcriptional regulator combinations, within a few days. dReaMGE adds to the repertoire of bacterial genome engineering to facilitate discovery, functional genomics, strain optimization and directed evolution of microbial cell factories.
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Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM... more
Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. ...
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Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but... more
Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformati...
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Sulfur is an essential element for all life by forming various sulfur-containing molecules. Compared to the well-studied biological functions, the knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule... more
Sulfur is an essential element for all life by forming various sulfur-containing molecules. Compared to the well-studied biological functions, the knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2- cd ]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes, and reconstitute the TPI skeleton in a one-pot enzymatic reaction. Particularly, we reveal that the JAMM/MPN + protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.
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Bacterial genomes encode numerous cryptic biosynthetic gene clusters (BGCs) that represent a largely untapped source of drugs or pesticides. Mining of the cryptic products is limited by the unavailability of streamlined genetic tools in... more
Bacterial genomes encode numerous cryptic biosynthetic gene clusters (BGCs) that represent a largely untapped source of drugs or pesticides. Mining of the cryptic products is limited by the unavailability of streamlined genetic tools in native producers. Precise genome engineering using bacteriophage recombinases is particularly useful for genome mining. However, recombinases are usually host-specific. The genome-guided discovery of novel recombinases and their transient expression could boost cryptic BGC mining. Herein, we reported a genetic system employing Red recombinases from Burkholderiales strain DSM 7029 for efficient genome engineering in several Burkholderiales species that currently lack effective genetic tools. Using specialized recombinases-assisted in situ insertion of functional promoters, we successfully mined five cryptic nonribosomal peptide synthetase/polyketide synthase BGCs, two of which were silent. Two classes of lipopeptides, glidopeptins and rhizomides, were...
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Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug... more
Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processe...
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The review highlights the 2013–2018 literature on the heterologous expression of bacterial natural product biosynthetic pathways and emphasises new techniques, heterologous hosts, and novel chemistry.
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A cryptic trans-acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide... more
A cryptic trans-acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide shuangdaolides A−D (1−4) and dumulmycin (5). Heterologous expression and gene inactivation experiments allowed the identification of two biosynthetic intermediates, 6 and 7, suggesting an unusual multidomain SDR oxidoreductase SdlR in charge of the formation of a rare 2-hydroxycyclopentenone moiety in this class of compounds.
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Genome mining of Fulvivirga sp. W222 revealed a desferrioxamine-like biosynthetic gene cluster containing an unknown gene f ulF that is conserved in many Bacteroidetes species. A series of primary amine-acylated desferrioxamine G 1... more
Genome mining of Fulvivirga sp. W222 revealed a desferrioxamine-like biosynthetic gene cluster containing an unknown gene f ulF that is conserved in many Bacteroidetes species. A series of primary amine-acylated desferrioxamine G 1 analogues, fulvivirgamides, were identified, and fulvivirgamides A 2 , B 2 , B 3 , and B 4 (1−4) were purified and characterized. The function of FulF, which is a novel acyltransferase for the acylation of the primary amine of Desferrioxamine G 1 , was verified by heterologous expression and feeding experiments.
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The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic... more
The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpress...
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The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product... more
The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A–H (1–8) and haereogladiodins A–B (9–10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1–8 and an E-Dhb in 9–10. Notably, compounds 2...
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Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were... more
Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS+ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential fo...
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Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in... more
Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, Pmwp , Pspc , PxylA , Pshuttle-09 , Pgrac or P43 , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. Th...
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In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine ((3-Acp)Ala) and 3-(trans-2’-nitrocyclopropyl)alanine ((3-Ncp)Ala) are originated via two homologous proteins BelK and HrmI, which... more
In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine ((3-Acp)Ala) and 3-(trans-2’-nitrocyclopropyl)alanine ((3-Ncp)Ala) are originated via two homologous proteins BelK and HrmI, which regioselectively catalyze Nε-nitration of L-lysine. The two enzymes belong to the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily and the catalytic center of BelK is validated by homology modeling and site-directed mutations. Based on the in vitro characterization, the biosynthetic pathways of (3-Acp)Ala and (3-Ncp)Ala are first proposed.
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Abstract Pseudomonas protegens CHA0 is a well-characterized plant-growth promoting rhizobacterium commonly used as biofertilizer or biocontrol agent for plant growth promotion and disease control. Genetically engineered strain... more
Abstract Pseudomonas protegens CHA0 is a well-characterized plant-growth promoting rhizobacterium commonly used as biofertilizer or biocontrol agent for plant growth promotion and disease control. Genetically engineered strain CHA0-ΔretS-nif with introduced nitrogen-fixing ability and enhanced bactericidal activity has the potential to stimulate plant growth and suppress phytopathogens. Field experiments was conducted to evaluate the effects of CHA0-ΔretS-nif inoculation on garlic growth, disease prevention and soil microbial diversity. Results revealed that CHA0-ΔretS-nif inoculation largely promoted garlic growth and yields. The total production of garlic in the CHA0-ΔretS-nif treated fields reached up to 34.25 T/ha, with an increase of 12.03% when compared to the untreated control (30.58 T/ha). When nitrogen application was reduced by 25%, CHA0-ΔretS-nif inoculation had a similar beneficial effects on garlic production with an increase of 10.62% than the uninoculated control. Moreover, CHA0-ΔretS-nif inoculation increased the bacterial population in the garlic rhizosphere and had significant effects against garlic root rot. Disease index in the CHA0-ΔretS-nif inoculated treatment (2.65%) was lower than the uninoculated control (3.31%). These results indicate that P. protegens CHA0-ΔretS-nif inoculation can help reduce chemical fertilizers and pesticides usage, and may be considered as an eco-friendly, viable alternative technology to increase food production in the future.
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Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH)... more
Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of ...
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Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01‐1 is a broad‐spectrum antagonistic bacterium present in plants. However, no... more
Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01‐1 is a broad‐spectrum antagonistic bacterium present in plants. However, no natural products have been isolated from this strain till date. Corresponding biosynthetic gene clusters to natural products is an effective method for bioprospecting, for which, genome manipulation tools are essential. We previously developed Pseudomonas‐specific phage‐derived homologous recombination systems for genetic engineering in four Pseudomonas species. Herein, we report the application of these recombineering systems in Pseudomonas parafulva CRS01‐1, along with structural elucidation and bioactivity evaluation of natural products. The Pseudomonas recombineering toolbox established before in different four species is efficient for genome mining and bioactive metabolite discovery from more distant species.
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Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical... more
Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative...
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The products of a nonribosomal peptide synthetase gene, holA, from Paraburkholderia rhizoxinica were investigated using our recently established recombineering technique. Fifteen products, including 13 new linear lipopeptides, holrhizins... more
The products of a nonribosomal peptide synthetase gene, holA, from Paraburkholderia rhizoxinica were investigated using our recently established recombineering technique. Fifteen products, including 13 new linear lipopeptides, holrhizins E-Q (2-8, 10-15), together with the two known holrhizins A and B (1, 9), were detected in the activated mutant, and their structures were identified using HRESIMS, NMR spectroscopy, Marfey's analysis, and feeding experiments with labeled amino acids. The lipohexapeptides 1-3 and 7-14 differ in three amino acid residues and the N-terminal fatty acid chains. The diversity of the holrhizins originates from the substrate flexibility of the A4, A5, and A6 domains as well as the starter C domain in the biosynthetic pathway.
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Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing... more
Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these ‘super bugs’. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell–cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs ...
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Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous... more
Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous recombination (HR)-based recombineering, mediated by the recombinases RecE/RecT from Rac prophage and Redα/Redβ from lambda phage, which has developed into a highly inclusive tool for direct cloning of large DNA up to 100 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs. Its utilization in the refactoring, engineering, and functional expression of long BGCs for NP biosynthesis makes it easy to elucidate NP-producing potential in microbes. This review also highlights various applications of recombineering in NP-derived drug discovery.
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Strain DSM 7029, isolated from a soil sample in Greece, can produce antitumour glidobactins, and has been found, as a heterologous host, to produce useful nonribosomal peptide synthetase-polyketide synthase hybrid molecules known as... more
Strain DSM 7029, isolated from a soil sample in Greece, can produce antitumour glidobactins, and has been found, as a heterologous host, to produce useful nonribosomal peptide synthetase-polyketide synthase hybrid molecules known as epothilones. This strain was originally named 'Polyangium brachysporum' of the family Polyangiaceae and the order Myxococcales. However, phylogenetic analysis of the 16S rRNA gene sequence of strain DSM 7029 indicated that it was clustered with members of Schlegelella. Significant growth occurred at 25-42 °C, pH 5.0-10.0 and in the presence of 0-0.2 % (w/v) NaCl. The predominant ubiquinone was Q-8. The major fatty acids were C16 : 1 ω7c/C16 : 1 ω6c, C16 : 0 and C18 : 1 ω7c. The G+C content of genomic DNA was 67.51 mol%. The strain was clearly distinguishable from other neighbouring Schlegelella members and genera Caldimonas and Zhizhongheella, using phylogenetic analysis, fatty acid composition data and a range of physiological and biochemical characteristics and genome analysis. Therefore, strain DSM 7029 represents a novel species of the genus Schlegelella, for which the name Schlegelella brevitalea sp. nov. is proposed.
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Abstract Widely used as drugs and agrochemicals, polyketides are a family of bioactive natural products, with diverse structures and functions. Polyketides are produced by megaenzymes termed as polyketide synthases (PKSs). PKS... more
Abstract Widely used as drugs and agrochemicals, polyketides are a family of bioactive natural products, with diverse structures and functions. Polyketides are produced by megaenzymes termed as polyketide synthases (PKSs). PKS biosynthetic pathways are divided into the cis-AT PKSs and trans-AT PKSs; a division based mainly on the absence of an acyltransferase (AT) domain in the trans-AT PKS modules. In trans-AT biosynthesis, the AT activity is contributed via one or several independent proteins, and there are few other characteristics that distinguish trans-AT PKSs from cis-AT PKSs, especially in the formation of the β-branch. The trans-AT PKSs constitute a major PKS pathway, and many are found in Burkholderia species, which are prevalent in the environment and prolific sources of polyketides. This review summarizes studies from 1973 to 2017 on the biosynthesis of natural products by trans-AT PKSs from Burkholderia species.
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Abnormal blood vessels and hypoxic and necrotic regions are common features of solid tumors and related to the malignant phenotype and therapy resistance. Certain obligate or facultative anaerobic bacteria exhibit inherent ability to... more
Abnormal blood vessels and hypoxic and necrotic regions are common features of solid tumors and related to the malignant phenotype and therapy resistance. Certain obligate or facultative anaerobic bacteria exhibit inherent ability to colonize and proliferate within solid tumors in vivo. Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic in European markets, has been known to proliferate selectively in the interface between the viable and necrotic regions of solid tumors. The objective of this study was to establish a tumor-targeting therapy system using the genetically engineered EcN for targeted delivery of cytotoxic compounds, including colibactin, glidobactin and luminmide. Biosynthetic gene clusters of these cytotoxic compounds were introduced into EcN and the corresponding compounds were detected in the resultant recombinant EcN strains. The recombinant EcN showed significant cytotoxic activity in vitro and in vivo as well, and significantly suppressed the tumor growth. Together, this study confirmed efficient tumor-targeting colonization of EcN and demonstrated its potentiality in the tumor-specific delivery of cytotoxic compounds as a new tumor-targeting therapy system.
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Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and... more
Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and Orf3, were modulated in the epothilone production strain Burkholderia DSM7029::Tn5-km-epo (named G32) via Red/ET engineering to increase heterologous polyketide epothilone yields. Compared with the prior G32 strain, the total production of several epothilones in the G32::orf14-orf3 mutant was meaningfully doubled according to high-performance liquid chromatography-mass spectrometer analysis. Typically for epothilone B, in simple and clear liquid medium CYMG, the overall productivity in the engineered high-yield producer G32::orf14-orf3 was improved for almost 3-fold, from 2.7 to about 8.1 μg/l. Additionally, the ratio of extracellular to intracellular accumulation of epothilone B was raised from 9.3:1 to 13.7:1 in response to expression of two putative...
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The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The... more
The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2- cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.
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Fluent genetic manipulation of prokaryote genomes is still limited to only a few commonly used hosts. Ideally the advanced technologies available for cloning into recombinant Escherichia coli should also be applicable in other... more
Fluent genetic manipulation of prokaryote genomes is still limited to only a few commonly used hosts. Ideally the advanced technologies available for cloning into recombinant Escherichia coli should also be applicable in other prokaryotes. In particular, 'recombineering' is mediated by the lambda Red operon that permits fluent and precise engineering of the E. coli genome and associated recombinant DNA. The major limitation is that host-specific phage-derived recombination systems are also required in more distant species. Recently, an endogenous Red-like operon Pluγβα has been reported to be effective in both Photorhabdus and Xenorhabdus bacteria. The Pluγβα recombineering system is based on three host-specific phage proteins from Photorhabdus luminescens, Plu2935, Plu2936, and Plu2934, which are functional analogs of Redβ, Redα, and Redγ, respectively. In this chapter, we provide a comprehensive and up-to-date method for P. luminescens and Xenorhabdus stockiae genome engineering via the Pluγβα recombineering system. In order to facilitate the rapid construction of knock-in vectors, recET-mediated recombineering is incorporated in the pipeline. Concerted recET system in E. coli with Pluγβα system in Photorhabdus and Xenorhabdus could promote reverse genetics, functional genomics, and bioprospecting research for these two genera.
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Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related... more
Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was suc...
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Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and... more
Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression w...
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Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol... more
Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The succe...
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Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural... more
Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural product salinomycin has a potent and selective activity against cancer stem cells and is therefore a potential anti-cancer drug. Herein, we separately isolated three fragments of the salinomycin gene cluster (salO-orf18) from Streptomyces albus (S. albus) DSM41398 using LLHR and assembled them into intact gene cluster (106 kb) by Red/ET and expressed it in the heterologous host Streptomyces coelicolor (S. coelicolor) A3(2). We are the first to report a large genomic region from a Gram-positive strain has been cloned using LLHR. The successful reconstitution and heterologous expression of the salinomycin gene cluster offer an attractive system for studying the function of the individual genes and identifying novel and potential analogues of complex natural...
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Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin... more
Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-D-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 ...