Daijie Chen

Thiopeptide Biosynthesis Featuring Ribosomally Synthesized Precursor Peptides and Conserved Posttranslational Modifications

Thiopeptides, with potent activity against various drug-resistant pathogens, contain a characteristic macrocyclic core consisting of multiple thiazoles, dehydroamino acids, and a 6-membered nitrogen heterocycle. Their biosynthetic pathways remain elusive, in spite of great efforts by in vivo feeding experiments. Here, cloning, sequencing, and characterization of the thiostrepton and siomycin A gene clusters unveiled a biosynthetic paradigm for the thiopeptide specific core formation, featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. The paradigm generality for thiopeptide biosynthesis was supported by genome mining and ultimate confirmation of the thiocillin I production in Bacillus cereus ATCC 14579, a strain that was previously unknown as a thiopeptide producer. These findings set the stage to accelerate the discovery of thiopeptides by prediction at the genetic level and to generate structural diversity by applying combinatorial biosynthesis methods.

Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production

β-Carotene is a terpenoid molecule with high hydrophobicity that is often used as an additive in foods and feed. Previous work has demonstrated the heterologous biosynthesis of β-carotene from an intrinsic high flux of acetyl-CoA in 12 steps through 11 genes in Yarrowia lipolytica. Here, an efficient biosynthetic pathway capable of producing 100-fold more β-carotene than the baseline construct was generated using strong promoters and multiple gene copies for each of the 12 steps. Using fed-batch fermentation with an optimized medium, the engineered pathway could produce 4g/L β-carotene, which was stored in lipid droplets within engineered Y. lipolytica cells. Expansion of these cells for squalene production also demonstrated that Y. lipolytica could be an industrially relevant platform for hydrophobic terpenoid production.

Whole cell microbial transformation in cloud point system

Cloud point system, consisting of nonionic surfactant in an aqueous solution, has been developed as a novel medium for whole cell microbial transformation. The basic properties of cloud point system including phase separation and solubilization are introduced. The application of cloud point system for extractive microbial transformation is different from that of water-organic solvent two-phase partitioning system or aqueous two-phase system are discussed, which mainly focus on the biocompatibility of microorganism in a cloud point system and a downstream process of microbial transformation in cloud point system with oil-water-surfactant microemulsion liquid-liquid extraction for surfactant recovery and product separation. Finally, examples of whole cell microbial transformation in cloud point systems, especially in situ extraction of moderate polar substrate/product, are also presented.

Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells

Oncogenic KRAS, an important target for antitumor therapy, contributes to pancreatic cancer tumorigenesis, progression and maintenance. However, intracellular compensation regulation can help cells to resist the targeted therapy. Gene knockdown method such as RNAi may help to understand this intracellular regulatory system and discover novel therapeutic approach. In this study, two stable transfected cell lines were constructed through lentivirus-mediated shRNA targeting KRAS of PANC-1 cells, to investigate cell response to the knockdown of KRAS. Human whole genome microarray was then used to compare the gene expression profile. As a result, ribosomal proteins L26 and L29 (RPL26 and RPL29) were dramatically upregulated by KRAS-shRNA specifically. To identify whether RPL26 or RPL29 was critical for PANC-1 cells, siRNAs against RPL26 and RPL29 were designed and transfected in vitro. The results showed that knockdown of RPL26 or RPL29 expression significantly suppressed cell proliferation, induced cell arrest at G0/G1 phase and enhanced cell apoptosis. Reactive oxygen species (ROS) assay indicated that silencing of RPL26 or RPL29 markedly decreased the intracellular ROS generation. Our findings imply that siRNA interference against RPL26 and RPL29 is of potential value for intervention of pancreatic cancer.

An Efficient and Novel Screening Model for Assessing the Bioactivity of Extracts against Multidrug-Resistant Pseudomonas aeruginosa Using Caenorhabditis elegans

As a large number of multidrug-resistant bacteria have emerged, and there is an urgent need for the development of new antibacterial agents. In this study, we developed a liquid-based slow killing assay to be carried out in standard 96-well microtiter plates. This screening method was designed to facilitate high-throughput screening of small molecules and extracts. In antibiotic rescue assays, the Caenorhabditis elegans multidrug-resistant Pseudomonas aeruginosa infection model displayed a high degree of drug resistance in vivo and in vitro. We used the method to screen 1,300 extracts, and found 36 extracts (2.7%) which prolonged the survival of infected nematodes, and four (0.3%) of these extracts showed in vitro and in vivo anti-multidrug resistant P. aeruginosa activity. These results indicate that the whole-animal C. elegans multidrug-resistant bacterial model can be used to screen antibacterial compounds, and can also be useful for bioactive compounds which most likely cannot be identified in vitro.

Screening of Endophytic Fungi with Antithrombotic Activity and Identification of a Bioactive Metabolite from the Endophytic Fungal Strain CPCC 480097

Fermentation extracts from 1,075 endophytic fungi were screened by the Markwardt method. The endophytic strain CPCC 480097 had the strongest antithrombotic activity and was identified as Fusarium sp. based on morphologic tests and internal transcriptional spacer sequence analysis. The target of the antithrombotic agent from the endophytic strain CPCC 480097 was identified by analysis of the fibrinogen clotting time, amidolytic activity, and fibrinolytic assay. The results showed that this antithrombotic agent is a 28-kDa single-chain fibrinolytic enzyme. The identification of this fibrinolytic enzyme was performed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The two internal sequences of this enzyme were obtained, and these showed no homology with those of other known fibrinolytic enzymes.

Microsphere resin chromatography combined with microbial biotransformation for the separation and purification of salvianolic acid B in aqueous extract of roots of Salvia multiorrihza Bunge.

Salvianolic acid B was separated and purified from Salvia miltiorrhiza Bunge (danshen) by microbial transformation together with chromatography of microsphere resin. The aqueous extract of danshen was transformed by Fusarium graminearum in a bioreactor containing phosphate buffer (PBS), in which rosmarinic acid was transformed into danshensu and caffeic acid and the yield of salvianolic acid B was higher than 85%. After biotransformation, salvianolic acid B was purified by microsphere resin. A parallel test for making a comparison of microsphere resin chromatography between elution by methanol water solution and water was done. The purity of salvianolic acid B was up to 95% at the yield of 62% when impurities and salvianolic acid B were eluted by 45% and 55% methanol solution respectively. The purity of salvianolic acid B was up to 99% at the yield of 90% when distilled water was used to elute the impurities and salvianolic acid B. The total yield of salvianolic acid B was up to 75% at the purity over 99% while biotransformation combined with microsphere resin chromatography by water elution. Microbial biotransformation together with water elution of microsphere resin supplied an efficient method to eliminate the micromolecular impurities and a possible method to purify water-soluble compounds in traditional Chinese medicine.

A novel phytotoxic nonenolide from Phomopsis sp. HCCB03520.

A novel phytotoxic nonenolide, (6S,7R,9R)‐6,7‐dihydroxy‐9‐propylnon‐4‐eno‐9‐lactone (1), was isolated from solid cultures of the endophytic fungus Phomopsis sp. HCCB03520, together with three known compounds, cytochalasin H (2), cytochalasin N (3), and epoxycytochalasin H (4). The structures of these compounds were elucidated through spectroscopic analysis, and the absolute configurations were determined by CD spectroscopy. Phytotoxic activities of compounds 1–4 were also investigated. Compound 1 showed phytotoxic activity on germination and radicle growth of Medicago sativa, Trifolium hybridum, and Buchloe dactyloides.

Efficient Bioconversion of Echinocandin B to Its Nucleus by Overexpression of Deacylase Genes in Different Host Strains

ABSTRACT Anidulafungin, which noncompetitively inhibits β-(1,3)-d-glucan synthase in fungal cell wall biosynthesis, is the newest antifungal drug to be developed. Echinocandin B deacylase from Actinoplanes utahensis NRRL 12052 catalyzes the cleavage of the linoleoyl group of echinocandin B, a key step in the process of manufacturing anidulafungin. Unfortunately, the natural yield of echinocandin B nucleus is low. In our study, the echinocandin B deacylase gene was systematically overexpressed by genetic engineering in its original producer, A. utahensis, and in the heterologous hosts Streptomyces lividans TK24 and Streptomyces albus. The introduction of additional copies of the gene, under the control of PermE* or its native promoter, into hosts showed significant increases in its transcription level and in the efficiency of the bioconversion of echinocandin B to its nucleus. The conditions for the cultivation and bioconversion of A. utahensis have been optimized further to improve production. As a result, while the wild-type strain initially produced 0.36 g/liter, a concentration of 4.21 g/liter was obtained after the generation of a strain with additional copies of the gene and further optimization of the reaction conditions. These results are useful for enhancing echinocandin B nucleus production in A. utahensis. Our study could enable the engineering of commercially useful echinocandin B nucleus-overproducing stains.

Overexpression of aveBIV leading to the improvement of 4'-epidaunorubicin production in Streptomyces coeruleorubidus strain SIPI-A0707.

The 4′-epidaunorubicin is the semisynthesis precursor of epirubicin which is a clinically useful antitumor drug thought to have slightly less cardiotoxicity than doxorubin. The 4′-epidaunorubicin was formed by overexpression of heterologous Streptomyces avermitilis aveBIV in Streptomyces coeruleorubidus SIPI-A0707 dnmV mutant blocked in the biosynthesis of daunosamine, the deoxysugar component of daunorubicin. But there was a low-yield production of 4′-epidaunorubicin. In our study, product yields were enhanced considerably by increasing aveBIV gene copy number or changing means of aveBIV integration. The 4′-epidaunorubicin titer was improved by around threefold in the recombinant strain DYG1006 with the aveBIV increased threefold copy numbers. The transcript levels of aveBIV gene meet the expectation of fermentation levels of 4′-epidaunorubicin. The method described here provides the means to produce 4′-epidaunorubicin efficiently on an industrial scale.