Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi.

Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5'-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.

Species-specificity of the secondary biosynthetic potential in Bacillus.

Introduction: Although Bacillus species have produced a wide variety of structurally diverse and biologically active natural products, the secondary biosynthetic potential of Bacillus species is widely underestimated due to the limited number of biosynthetic gene clusters (BGCs) in this genus. The significant variation in the diversity and novelty of BGCs across different species within the Bacillus genus presents a major obstacle to the efficient discovery of novel natural products from Bacillus. Methods: In this study, the number of each class of BGCs in all 6,378 high-quality Bacillus genomes was predicted using antiSMASH, the species-specificity of BGC distribution in Bacillus was investigated by Principal component analysis. Then the structural diversity and novelty of the predicted secondary metabolites in Bacillus species with specific BGC distributions were analyzed using molecular networking. Results: Our results revealed a certain degree of species-specificity in the distribution of BGCs in Bacillus, which was mainly contributed by siderophore, type III polyketide synthase (T3PKS), and transAT-PKS BGCs. B. wiedmannii, B. thuringiensis, and B. cereus are rich in RiPP-like and siderophore BGCs, but lack T3PKS BGCs, while B. amyloliquefaciens and B. velezensis are abundant in transAT-PKS BGCs. These Bacillus species collectively encode 77,541 BGCs, with NRPS and RiPPs being the two most dominant types, which are further categorized into 4,291 GCFs. Remarkably, approximately 54.5% of GCFs and 93.8% of the predicted metabolite scaffolds are found exclusively in a single Bacillus species. Notably, B. cereus, B. thuringiensis, and B. velezensis exhibit the highest potential for producing species-specific NRPS and PKS bioinformatic natural products. Taking two species-specific NRPS gene clusters as examples, the potential of Bacillus to synthesize novel species-specific natural products is illustrated. Conclusion: This study highlights the species-specificity of the secondary biosynthetic potential in Bacillus and provides valuable insights for the targeted discovery of novel natural products from this genus.

Global analysis of the biosynthetic chemical space of marine prokaryotes.

BACKGROUND: Marine prokaryotes are a rich source of novel bioactive secondary metabolites for drug discovery. Recent genome mining studies have revealed their great potential to bio-synthesize novel secondary metabolites. However, the exact biosynthetic chemical space encoded by the marine prokaryotes has yet to be systematically evaluated. RESULTS: We first investigated the secondary metabolic potential of marine prokaryotes by analyzing the diversity and novelty of the biosynthetic gene clusters (BGCs) in 7541 prokaryotic genomes from cultivated and single cells, along with 26,363 newly assembled medium-to-high-quality genomes from marine environmental samples. To quantitatively evaluate the unexplored biosynthetic chemical space of marine prokaryotes, the clustering thresholds for constructing the biosynthetic gene cluster and molecular networks were optimized to reach a similar level of the chemical similarity between the gene cluster family (GCF)-encoded metabolites and molecular family (MF) scaffolds using the MIBiG database. The global genome mining analysis demonstrated that the predicted 70,011 BGCs were organized into 24,536 mostly new (99.5%) GCFs, while the reported marine prokaryotic natural products were only classified into 778 MFs at the optimized clustering thresholds. The number of MF scaffolds is only 3.2% of the number of GCF-encoded scaffolds, suggesting that at least 96.8% of the secondary metabolic potential in marine prokaryotes is untapped. The unexplored biosynthetic chemical space of marine prokaryotes was illustrated by the 88 potential novel antimicrobial peptides encoded by ribosomally synthesized and post-translationally modified peptide BGCs. Furthermore, a sea-water-derived Aquimarina strain was selected to illustrate the diverse biosynthetic chemical space through untargeted metabolomics and genomics approaches, which identified the potential biosynthetic pathways of a group of novel polyketides and two known compounds (didemnilactone B and macrolactin A 15-ketone). CONCLUSIONS: The present bioinformatics and cheminformatics analyses highlight the promising potential to explore the biosynthetic chemical diversity of marine prokaryotes and provide valuable knowledge for the targeted discovery and biosynthesis of novel marine prokaryotic natural products. Video Abstract.

RETRACTED ARTICLE: Mutagenesis combined with fermentation optimization to enhance gibberellic acid GA3 yield in Fusarium fujikuroi.

Gibberellic acid (GA3) is a plant growth hormone that plays an important role in the production of crops, fruits, and vegetables with a wide market share. Due to intrinsic advantages, liquid fermentation of Fusarium fujikuroi has become the sole method for industrial GA3 production, but the broader application of GA3 is hindered by low titer. In this study, we combined atmospheric and room-temperature plasma (ARTP) with ketoconazole-based screening to obtain the mutant strain 3-6-1 with high yield of GA3. Subsequently, the medium composition and fermentation parameters were systematically optimized to increase the titer of GA3, resulting in a 2.5-fold increase compared with the titer obtained under the initial conditions. Finally, considering that the strain is prone to substrate inhibition and glucose repression, a new strategy of fed-batch fermentation was adopted to increase the titer of GA3 to 575.13 mg/L, which was 13.86% higher than the control. The strategy of random mutagenesis combined with selection and fermentation optimization developed in this study provides a basis for subsequent research on the industrial production of GA3.

Microbacterium karelineae sp. nov. isolated from a halophyte plant in the Taklamakan desert.

A bacterial strain, designated TRM 80801T, was isolated from the Karelinea in Taklamakan desert, Xinjiang Uygur Autonomous Region, north-west China. Cells were Gram-stain-positive, aerobic, non-motile, short rods. Strain TRM 80801T grew at 4-50 °C, with optimum growth at 28 °C, and grew at pH 6.0-11.0 and 1-15 % (w/v) NaCl. Phylogenetic analyses of the 16S rRNA gene sequences placed strain TRM 80801T within the genus Microbacterium with the highest similarities to Microbacterium suaedae YZYP 306T (98.97 %) and Microbacterium indicum BBH6T (98.17 %), respectively. The DNA G+C content of TRM 80801T is 69.38 mol%. The cell-wall peptidoglycan contained the amino acids ornithine, glutamic acid, glycine and alanine, the diagnostic diamino acid was ornithine. The acyl type of the peptidoglycan was glycolyl. Whole-cell sugars were ribose, mannose, glucose, rhamnose and galactose. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The predominant menaquinones were MK-10, MK-11 and MK-12. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol. The whole-genome average nucleotide identity (ANI) value between strain TRM 80801T and Microbacterium suaedae YZYP 306T is 70.2 %. On the basis of the evidence presented in this study, strain TRM 80801T is representative of a novel species in the genus Microbacterium, for which the name Microbacterium karelineae sp. nov. is proposed. The type strain is TRM 80801T (=CCTCC AB 2019248T=KCTC 49357T).

YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica.

Non-homologous end-joining (NHEJ)-mediated random integration in Yarrowia lipolytica has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Y. lipolytica. By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.

New pyrazinoquinazoline alkaloids Isolated from a culture of Stenotrophomonas maltophilia QB-77.

Two new pyrazinoquinazoline alkaloids, epi-fiscalin D (1) and epi-fiscalin E (2), as well as three known analogues, norquinadoline A (3), quinadoline A (4), and fiscalin C (5), were isolated from ethyl acetate extract of the fermentation broth of Stentrophomonas maltophilia QB-77. The structures of new compounds were elucidated on the basis of extensive spectroscopic data analysis including UV, HRESIMS, and 1D and 2D NMR experiments. All the isolated compounds were tested for their in vitro cytotoxicity against five human cancer cell lines (SMMC-7721, MCF-7, HL-60, SW480, and A-549) and antibacterial activities against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.

The changes and its significance of Th17 and Treg cells and related cytokines in patients with tuberculosis pleurisy.

BACKGROUND: Tuberculous pleurisy is a kind of tuberculosis, it is well known that Th1 lymphocytes play a key role in the treatment of tuberculosis infection. However, latest studies show that Th17 lymphocyte may also play an important role tuberculosis infection. There is close relationship between Treg and Thl7 cells, and changes in the number or the function of the two kinds of cells may lead to diseases. The current researches on Thl7 and Treg cells maily focus on autoimmune diseases, however, reports about their role in tuberculosis are limited. In this study, we investigate the function of th17 and Treg cells and the above cytokines in the pathogenesis of tuberculosis pleurisy; by determining the expression of Th17 and Treg cells in peripheral CD4 T cells and the related cytokines in patients with tuberculous compared with healthy people. RESULTS: Th17 cells in patients were higher than that in the Healthy control group, expression of Treg cells in patients were lower than that in the healthy group; IL-17, IL-23 levels in peripheral blood and hydrothorax from the patients were higher than that in the healthy group; IL-17, IL-23 and IL-6 levels in hydrothorax were higher than that in peripheral blood. There was no difference in IL-6 level in peripheral blood between the patients and healthy control; TGF- ß level in peripheral blood from the healthy group was higher than that in peripheral blood and hydrothorax from the patients. And there were no differences in TGF- ß level between peripheral blood and hydrothorax. Th17 cells were negatively correlated with Treg cells ,but were positive correlation with IL-17, IL-23, IL-6 levels in peripheral blood; TGF- ß level was positive correlation with Treg cells in the peripheral blood, but no correlation with Th17 cells. CONCLUSION: Th17 and Treg cells may be involved in the immune pathological mechanism of tuberculous pleurisy and changes of related cytokines may be involved in the differentiation of Th17 and Treg cells and inflammatory response. Thus, Th17 and Treg cells and related cytokines may be important immunopathogenesis for tuberculous pleurisy.