Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

AIMS: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. METHODS AND RESULTS: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l). CONCLUSIONS: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.

[Weidiao-3 Mixture Improves the Clinical Efficacy of Immunotherapy for Advanced Gastric Cancer by Regulating Intestinal Flora].

Objective To investigate the effects of Weidiao-3(WD-3)Mixture on the clinical efficacy of immunotherapy for advanced gastric cancer and the intestinal flora.Methods Fifty-one patients with advanced gastric cancer treated in Wuxi Traditional Chinese Medicine Hospital from January 2020 to December 2021 were randomized into a WD-3 group(immunotherapy + WD-3 Mixture,one dose per day)(n=25)and a gastric cancer(GC) group(only immunotherapy)(n=26)according to the admission time.Ten healthy volunteers were included as the healthy control group.The Karnofsky score and the Quality of Life Questionnare-Core score were evaluated before and after treatment,and the clinical efficacy was compared after treatment.After treatment,the stool samples were collected for 16SrRNA gene high-throughput sequencing and targeted metabolomics.The α and ß diversity and structure of the intestinal flora and the content of short-chain fatty acids were compared between groups.Results The quality of life in both groups improved after treatment and was better in the WD-3 group than in the GC group(P=0.035).The dry mouth(P=0.038)and altered taste(P=0.008)were mitigated in the WD-3 group after treatment,and the reflux(P=0.001)and dry mouth(P=0.022)were mitigated in the GC group after treatment.After treatment,the WD-3 group outperformed the GC group in terms of dysphagia(P=0.047)and dry mouth(P=0.045).The WD-3 group was superior to the GC group in terms of objective remission rate and disease control rate,with prolonged median progression-free survival and median overall survival(P=0.039,P=0.043).The α and ß diversity indexes of the intestinal flora showed no significant differences between WD-3 and GC groups(all P>0.05).At the phylum level,WD-3 and GC groups had lower relative abundance of Firmicutes(P=0.038,P=0.042)and higher relative abundance of Proteobacteria(P=0.016,P=0.015)than the healthy control group.The relative abundance of Actinomycetes in the GC group was lower than that in the healthy control group(P=0.035)and the WD-3 group(P=0.046).At the genus level,the GC group had lower relative abundance of Bifidobacteria and Coprococcus than the healthy control group and the WD-3 group(all P<0.001).LEfSe revealed the differences in the relative abundance of 6 intestinal bacterial taxa between the WD-3 group and the GC group.At the genus level,Saccharopolyspora had higher relative abundance in the WD-3 group than in the healthy control group and only existed in the WD-3 group.The content of isobutyric acid and isovaleric acid in the WD-3 group was higher than that in the healthy control group(P=0.037,P=0.004).Conclusion WD-3 Mixture may increase the relative abundance of Bifidobacteria and Coprococcus and the content of isobutyric acid and isovaleric acid to alter the intestinal microecology,thereby improving the efficacy of immunotherapy for gastric cancer.

Identification and Characterization of a New Regulator, TagR, for Environmental Stress Resistance Based on the DNA Methylome of Streptomyces roseosporus.

DNA methylation is a defense that microorganisms use against extreme environmental stress, and improving resistance against environmental stress is essential for industrial actinomycetes. However, research on strain optimization utilizing DNA methylation for breakthroughs is rare. Based on DNA methylome analysis and KEGG pathway assignment in Streptomyces roseosporus, we discovered an environmental stress resistance regulator, TagR. A series of in vivo and in vitro experiments identified TagR as a negative regulator, and it is the first reported regulator of the wall teichoic acid (WTA) ABC transport system. Further study showed that TagR had a positive self-regulatory loop and m4C methylation in the promoter improved its expression. The ΔtagR mutant exhibited better hyperosmotic resistance and higher decanoic acid tolerance than the wild type, which led to a 100% increase in the yield of daptomycin. Moreover, enhancing the expression of the WTA transporter resulted in better osmotic stress resistance in Streptomyces lividans TK24, indicating the potential for wide application of the TagR-WTA transporter regulatory pathway. This research confirmed the feasibility and effectiveness of mining regulators of environmental stress resistance based on the DNA methylome, characterized the mechanism of TagR, and improved the resistance and daptomycin yield of strains. Furthermore, this research provides a new perspective on the optimization of industrial actinomycetes. IMPORTANCE This study established a novel strategy for screening regulators of environmental stress resistance based on the DNA methylome and discovered a new regulator, TagR. The TagR-WTA transporter regulatory pathway improved the resistance and antibiotic yield of strains and has the potential for wide application. Our research provides a new perspective on the optimization and reconstruction of industrial actinomycetes.

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Improving the Yield and Quality of Daptomycin in Streptomyces roseosporus by Multilevel Metabolic Engineering.

Daptomycin is a cyclic lipopeptide antibiotic with a significant antibacterial action against antibiotic-resistant Gram-positive bacteria. Despite numerous attempts to enhance daptomycin yield throughout the years, the production remains unsatisfactory. This study reports the application of multilevel metabolic engineering strategies in Streptomyces roseosporus to reconstruct high-quality daptomycin overproducing strain L2797-VHb, including precursor engineering (i.e., refactoring kynurenine pathway), regulatory pathway reconstruction (i.e., knocking out negative regulatory genes arpA and phaR), byproduct engineering (i.e., removing pigment), multicopy biosynthetic gene cluster (BGC), and fermentation process engineering (i.e., enhancing O2 supply). The daptomycin titer of L2797-VHb arrived at 113 mg/l with 565% higher comparing the starting strain L2790 (17 mg/l) in shake flasks and was further increased to 786 mg/l in 15 L fermenter. This multilevel metabolic engineering method not only effectively increases daptomycin production, but can also be applied to enhance antibiotic production in other industrial strains.

A novel strategy of gene screen based on multi-omics in Streptomyces roseosporus.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.

[Procalcitonin impact analysis, respiratory function and blood gas analysis on Xiaoqinglong decoction combined with non-invasive ventilation in treatment of AECOPD patients].

To observe the effect of Xiaoqinglong decoction combined with noninvasive ventilation on procalcitonin (PCT), blood gas analysis and respiratory functions in acute exacerbation of chronic obstructive pulmonary disease in the elderly (AECOPD), and investigate its correlation and clinical significance. Eighty-four elderly AECOPD patients with respiratory failure in our hospital from January 2015 to October 2017, were randomly divided into control group and observation group, 42 cases in each group. The control group received western medicine combined with noninvasive ventilator therapy, and the patients in observation group additionally received Xiaoqinglong decoction on the basis of the treatment in control group. Both groups were treated for 2 weeks. The clinical effects of two groups were observed and their PCT, blood gas analysis outcomes [arterial oxygen partial pressure (PaO2), arterial partial pressure of carbon dioxide (PaCO2), respiratory function, forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), FEV1/FVC], TCM syndrome score and other indexes and adverse reactions were compared before and after treatment. The total efficiency was 95.24% (40/42) in observation group, higher than 76.19% (32/42) in control group, with statistically significant difference (P<0.05). There were no statistically significant differences in PCT, PaO2, PaCO2, FVC, FEV1/FVC, FEV1, and TCM syndrome scores between two groups before treatment. But after treatment, PCT and PaCO2 levels in the observation group were lower and PaO2, FVC, FEV1/FVC, FEV1 levels was higher than those in the control group (P<0.05); TCM syndrome scores were lower than those in the control group (P<0.05); both groups had no obvious adverse reactions. The results showed that Xiaoqinglong decoction combined with noninvasive ventilator could significantly reduce the procalcitonin level, effectively improve the respiratory function and blood gas analysis indexes, and significantly reduce the clinical symptoms in AECOPD patients, so it is worthy of promotion.

[Contribution of Nitrogen Sources in Water Sources by Combining Nitrogen and Oxygen Isotopes and SIAR].

It is very important to identify nitrate sources in reservoirs that serve as high quality water sources to control its eutrophication. Stable isotopes (δ15 N and δ18O) and a Bayesian model (stable isotope analysis in R, SIAR) were applied to identify nitrate sources and estimate the proportional contributions of multiple nitrate sources in four reservoirs (Qingshan reservoir, Duihekou reservoir, Siling reservoir, and Lifan reservoir) that serve as sources of drinking water in the Hangjiahu area, one of the most densely populated and most quickly developing areas in East China. It was shown that nitrogen pollution, which was dominated by nitrate (NO3-), existed in the four reservoirs. Greater human activities caused more nitrogen pollution (average NO3- concentration 0.21 mmol ·L-1) in the Qingshan reservoir. A significant positive correlation (P<0.01) was observed between Cl- and NO3-. The analysis of the water in the Duihekou reservoir, Siling reservoir, and Lifan reservoir, with lower Cl- concentrations and higher NO3-/Cl- ratios, suggested that chemical fertilizer was the main source, while the analysis of the water in the Qingshan reservoir, with medium Cl- concentrations and NO3-/Cl- ratios, indicated a mixture of NO3- sources. The δ15 N ranged from 0.9‰ to 7.2‰, and the δ18O ranged from 2.8‰ to 14.1‰ in the four reservoirs. The δ18O values in more than 86% of the water samples were less than 10‰, and the δ15 N/δ18O values in 93% of the water samples were less than 1.3. It was identified that nitrification rather than denitrification acted as the primary N cycling process in the four reservoirs. SIAR was used to estimate the proportional contribution of five NO3- sources (industrial wastewater, sewage/manure, chemical fertilizer, soil nitrogen, and precipitation) in the Qingshan reservoir and of three NO3- sources (chemical fertilizer, soil nitrogen, and precipitation) in the Duihekou reservoir, Siling reservoir, and Lifan reservoir. The source apportionment results showed that chemical fertilizers and soil nitrogen were the dominant nitrate sources and their contributions were 75%-82%. It was revealed that nitrogen pollution in the water source reservoir caused by cropping non-point source pollution was very serious. Nitrate source contributions in Qingshan reservoir also included sewage/manure (25%), soil nitrogen (7%), and precipitation (6%), indicating that nitrogen pollution by sewage/manure should not be ignored in the higher human activity areas. The nitrate source in the Duihekou reservoir, Siling reservoir, and Lifan reservoir also included precipitation, with the nitrate contribution from precipitation at 21%, 24%, and 15%, respectively. It was suggested that precipitation contributed more nitrate to the water in areas with less human activity.