Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Discovery and Biosynthetic Studies of a Highly Reduced Cinnamoyl Lipid, Tripmycin A, from an Endophytic Streptomyces sp.

A Tripterygium wilfordii endophyte, Streptomyces sp. CB04723, was shown to produce an unusually highly reduced cytotoxic cinnamoyl lipid, tripmycin A (1). Structure-activity relationship studies revealed that both the cinnamyl moiety and the saturated fatty acid side chain are indispensable to the over 400-fold cytotoxicity improvement of 1 against the triple-negative breast cancer cell line MDA-MB-231 compared to 5-(2-methylphenyl)-4-pentenoic acid (2). Bioinformatical analysis, gene inactivation, and overexpression revealed that Hxs15 most likely acted as an enoyl reductase and was involved with the side chain reduction of 1, which provides a new insight into the biosynthesis of cinnamoyl lipids.

Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A.

Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0 mg/L TNM-A in shaking flasks and 20.8 ± 0.4 mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8 kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.Key Points •United genome shuffling and ribosome engineering help further strain improvement. •CB03234-GS26 with improved titer is practical for the pilot production of TNM-A. •Enhanced TNM-A production should attribute to novel genetic features/mechanisms.

Recycling of Chinese herb residues by endophytic and probiotic fungus Aspergillus cristatus CB10002 for the production of medicinal valuable anthraquinones.

BACKGROUND: The global prevalence of traditional Chinese medicine stimulates the prosperous development of herb medicines, but the annual generation of massive herb residues becomes big issues about environmental pollution and waste of resources. Microbes play important roles in the circulation of substances in nature, and endophytes represent an underexplored microbial resource possessing the unique symbiotic relationship with plants, not only for discovery of secondary metabolites, but also for potential green recycling of herb residues. RESULTS: The recycling capacities of several endophytic strains were respectively evaluated via solid state fermentation with herb residues of commercial Huazhenghuisheng oral-liquid (HOL). Among them, Aspergillus cristatus CB10002, a probiotic fungus isolated from Chinese Fu-brick tea, was competent to recycle HOL residues for the production of medicinal valuable anthraquinones, in which four of them, especially citreorosein with significant anti-obesity activity, were first discovered in A. cristatus. Subsequent quantitative analysis showed that about 2.0 mg/g citreorosein and 7.5 mg/g total anthraquinones could be obtained after 35-day fermentation, which was very competitive and economically beneficial. Further nutritional comparisons also revealed that the recycling process indeed ameliorated the nutrients of HOL residues, and thus proposed a possibility to directly dispose the final leftovers as a compost organic fertilizer. CONCLUSIONS: The endophytic and probiotic fungus A. cristatus CB10002 isolated from Chinese Fu-brick tea was screened out to effectively reutilize HOL residues for the production of nine medicinal valuable anthraquinones, whose biosynthesis may be regulated by the induction of HOL residues. The competitive yields of these anthraquinones, as well as the certain composting properties of final leftovers, have made the microbial recycling of HOL residues economically beneficial. Our work demonstrated a promising applied potential of A. cristatus in reutilization of herb residues, and provided a practical strategy for sustainable and value-added microbial recycling of herb residues.

Effect of Polyethylene Glycol on the NiO Photocathode.

In this study, a uniform nanoporous NiO film, with a thickness of up to 2.6 µm, was prepared using polyethylene glycol (PEG). The addition of PEG significantly decreased the cracks in the NiO film and prevented the peeling of the NiO film from a fluorine-doped tin oxide substrate. The NiO cathode was prepared using CdSeS quantum dots (QDs) as the sensitizer, with an optimized photoelectric conversion of 0.80%. The optimized QD-sensitized NiO films were first assembled with the TiO2 anode to prepared QD-sensitized p-n-type tandem solar cells. The open circuit voltage was greater than that obtained using the separated NiO cathode or TiO2 anode.

CdS/CdSe Co-sensitized Solar Cells Based on Hierarchically Structured SnO2/TiO2 Hybrid Films.

SnO2 nanosheet-structured films were prepared on a fluorine-doped tin oxide (FTO) substrate using ZnO nanosheet as template. The as-prepared SnO2 nanosheets contained plenty of nano-voids and were generally vertical to the substrate. TiO2 nanoparticles were homogeneously deposited into the intervals between the SnO2 nanosheets to prepare a hierarchically structured SnO2/TiO2 hybrid film. The hybrid films were co-sensitized with CdS and CdSe quantum dots. The sensitized solar cells assembled with the SnO2/TiO2 hybrid film showed much higher photoelectricity conversion efficiency than the cells assembled with pure TiO2 films. The lifetime of photoinduced electron was also investigated through electrochemical impedance spectroscopy, which showed that the SnO2/TiO2 hybrid film electrode is as long as the TiO2 film electrode.