High-yield production of FK228 and new derivatives in a Burkholderia chassis.

FK228 (romidepsin) is the only natural histone deacetylases (HDACs) inhibitor approved by FDA to treat cutaneous and peripheral T-cell lymphoma. However, the limited supply and severe cardiotoxicity of FK228 underscore the importance to develop an effective synthetic biology platform for the manufacturing and fine-tuning of this drug lead. In this work, we constructed a Burkholderia chassis for the high-yield production of FK228-family (unnatural) natural products. By virtue of the optimized Burkholderia-specific recombineering system, the biosynthetic gene cluster (BGC) encoding the FK228-like skeleton thailandepsins (tdp) in Burkholderia thailandensis E264 was replaced with an attB integration site to afford the basal chassis KOGC1. The tdp BGC directly captured from E264 was hybridized with the FK228-encoding BGC (dep) using the versatile Red/ET technology. The hybrid BGC (tdp-dep) was integrated into the attB site of KOGC1, resulting in the heterologous expression of FK228. Remarkably, the titer reached 581 mg/L, which is 30-fold higher than that of native producer Chromobacterium violaceum No. 968. This success encouraged us to further engineer the NRPS modules 4 or 6 of hybrid tdp-dep BGC by domain units swapping strategy, and eight new FK228 derivatives (1-8) varying in the composition of amino acids were generated. Especially, the titers of 2 and 3 in KOGC1 were up to 985 mg/L and 453 mg/L, respectively. 2 and 3 displayed stronger cytotoxic activity than FK228. All in all, this work established a robust platform to produce FK228 and its new derivatives in sufficient quantities for anticancer drug development.

Streptomyces: The biofactory of secondary metabolites.

Natural products derived from microorganisms serve as a vital resource of valuable pharmaceuticals and therapeutic agents. Streptomyces is the most ubiquitous bacterial genus in the environments with prolific capability to produce diverse and valuable natural products with significant biological activities in medicine, environments, food industries, and agronomy sectors. However, many natural products remain unexplored among Streptomyces. It is exigent to develop novel antibiotics, agrochemicals, anticancer medicines, etc., due to the fast growth in resistance to antibiotics, cancer chemotherapeutics, and pesticides. This review article focused the natural products secreted by Streptomyces and their function and importance in curing diseases and agriculture. Moreover, it discussed genomic-driven drug discovery strategies and also gave a future perspective for drug development from the Streptomyces.

Efficacy of recombinant Newcastle disease virus expressing HA protein of H9N2 Avian influenza virus in respiratory and intestinal tract.

H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV, which is widely prevalent all over the world. The infection of H9N2 AIV often leads to secondary infection with other pathogens, causing serious economic losses to poultry industry. Up to now, several recombinant Newcastle disease viruses (NDV) expressing H9N2 AIV hemagglutinin (HA) protein had been developed. However, the efficacy of recombinant virus on tracheal and intestinal injury caused by H9N2 AIV was rarely reported. The aim of this study was to evaluate the efficacy of recombinant NDV expressing H9N2 AIV HA protein in respiratory and intestinal tract. In this study, based on Red/ET homologous recombination technology, H9N2 AIV HA gene was embedded into the genome of NDV LaSota vaccine strain to obtain the recombinant virus rNDV-H9. The recombinant virus rNDV-H9 showed similar replication kinetic characteristics with the parent LaSota strain and had good genetic stability. The immunization result showed that rNDV-H9 induced high HI antibody titer against H9N2 AIV. In the H9N2 AIV challenge experiment, rNDV-H9 could significantly reduce the virus shedding in trachea and cloaca. In addition, rNDV-H9 protected the barrier function of chicken intestinal mucosal epithelial cells and reduced the virus-induced inflammatory response to a certain extent, so as to inhibit the abnormal proliferation of E. coli. This study suggests that rNDV-H9 is a promising vaccine candidate against H9N2 AIV.

Establishing an efficient salinomycin biosynthetic pathway in three heterologous Streptomyces hosts by constructing a 106-kb multioperon artificial gene cluster.

Salinomycin is a promising anticancer drug for chemotherapy. A highly productive biosynthetic gene cluster will facilitate the creation of analogs with improved therapeutic activity and reduced side effects. In this study, we engineered an artificial 106-kb salinomycin gene cluster and achieved efficient heterologous expression in three hosts: Streptomyces coelicolor CH999, S. lividans K4-114, and S. albus J1074. The six-operon artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid ß-oxidation genes into one operon. All operons are driven by strong constitutive promoters. For K4-114 and J1074 harboring the artificial gene cluster, salinomycin production in shake flask cultures was 14.3 mg L-1 and 19.3 mg L-1 , respectively. The production was 1.3-fold and 1.7-fold higher, respectively, than that of the native producer S. albus DSM41398. K4-114 and J1074 harboring the native gene cluster produced an undetectable amount of salinomycin and 0.5 mg L-1 , respectively. CH999 harboring the artificial gene cluster produced 10.3 mg L-1 of salinomycin, which was 92% of the production by DSM41398. The efficient heterologous expression system based on the 106-kb multioperon artificial gene cluster established in this study will facilitate structural diversification of salinomycin, which is valuable for drug development and structure-activity studies.

Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii.

The lambda phage Red proteins Redα/Redß/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecETheBDU8 , RecEThTJI49 and RecETh1h2eYI23 , which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecEThTJI49 and RecETh1h2eYI23 showed higher recombination efficiency compared to RecETheBDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2eYI23, RecEThTJI49 and RecETheBDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2eYI23 . Additionally, RecETYI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγßα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαß mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3'-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3'-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

Enhanced Heterologous Spinosad Production from a 79-kb Synthetic Multioperon Assembly.

Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.