Development of potent and broad-spectrum drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains one of the top priorities, especially in the cases of the emergence of mutant viruses and inability of current vaccines to prevent viral transmission. In this study, we have generated a novel membrane fusion-inhibitory lipopeptide IPB29, which is currently under clinical trials; herein, we report its design strategy and preclinical data. First, we surprisingly found that IPB29 with a rigid linker between the peptide sequence and lipid molecule had greatly improved α-helical structure and antiviral activity. Second, IPB29 potently inhibited a large panel of SARS-CoV-2 variants including the previously and currently circulating viruses, such as Omicron XBB.5.1 and EG.5.1. Third, IPB29 could also cross-neutralize the bat- and pangolin-isolated SARS-CoV-2-related CoVs (RatG13, PCoV-GD, and PCoV-GX) and other human CoVs (SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E). Fourth, IPB29 administrated as an inhalation solution (IPB29-IS) in Syrian hamsters exhibited high therapeutic and preventive efficacies against SARS-CoV-2 Delta or Omicron variant. Fifth, the pharmacokinetic profiles and safety pharmacology of IPB29-IS were extensively characterized, providing data to support its evaluation in humans. In conclusion, our studies have demonstrated a novel design strategy for viral fusion inhibitors and offered an ideal drug candidate against SARS-CoV-2 and other coronaviruses.

Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, a cyclic lipopeptide with a C12-C17 β-hydroxy fatty acid chain, exhibits significant antibacterial activity and resists resistance, making it a research focus. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin\'s structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by Bacillus velezensis YA215. Process optimization shows that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximized surfactin production to 18.59%, 19.23%, and 20.64%, respectively. Surfactin content peaked at 36 h with L-Val and L-Ile, yielding 19.72% and 11.37%. In contrast, L-Leu addition peaked at 24 h, yielding 11.33%. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia coli and Staphylococcus aureus, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 β-hydroxy fatty acids from 13.3% and 4.216-23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a \"last-line antibiotic\", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

The incorporation of well-designed antibiotic nanocarriers, along with an antibiotic adjuvant effect, in combination with various antibiotics, offers an opportunity to combat drug-resistant strains. However, precise control over morphology and encapsulated payload release can significantly impact their antibacterial efficacy and synergistic effects when used alongside antibiotics. Here, this study focuses on developing lipopeptide-based nanoantibiotics, which demonstrate an antibiotic adjuvant effect by inducing pH-induced collapse and negative-charged-surface-induced deformation. This enhances the disruption of the bacterial outer membrane and facilitates drug penetration, effectively boosting the antimicrobial activity against drug-resistant strains. The modulation regulations of the lipopeptide nanocarriers with modular design are governed by the authors. The nanoantibiotics, made from lipopeptide and ciprofloxacin (Cip), have a drug loading efficiency of over 80%. The combination with Cip results in a significantly low fractional inhibitory concentration index of 0.375 and a remarkable reduction in the minimum inhibitory concentration of Cip against multidrug-resistant (MDR) Escherichia coli (clinical isolated strains) by up to 32-fold. The survival rate of MDR E. coli peritonitis treated with nanoantibiotics is significantly higher, reaching over 87%, compared to only 25% for Cip and no survival for the control group. Meanwhile, the nanoantibiotic shows no obvious toxicity to major organs.

Fengycin is an important member of the lipopeptide family with a wide range of applications in the agricultural, food, medical and cosmetic industries. However, its commercial application is severely hindered by low productivity and high cost. Therefore, numerous studies have been devoted to improving the production of fengycin. We summarize these studies in this review with the aim of providing a reference and guidance for future researchers. This review begins with an overview of the synthesis mechanism of fengycin via the non-ribosomal peptide synthetases (NRPS), and then delves into the strategies for improving the fengycin production in recent years. These strategies mainly include fermentation optimization and metabolic engineering, and the metabolic engineering encompasses enhancement of precursor supply, application of regulatory factors, promoter engineering, and application of genome-engineering (genome shuffling and genome-scale metabolic network model). Finally, we conclude this review with a prospect of fengycin production.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

Fusarium wilt is a worldwide soil-borne fungal disease caused by Fusarium oxysporum that causes serious damage to agricultural products. Therefore, preventing and treating fusarium wilt is of great significance. In this study, we purified ten single lipopeptide fengycin components from Bacillus subtilis FAJT-4 and found that C17 fengycin B inhibited the growth of F. oxysporum FJAT-31362. We observed early apoptosis hallmarks, including reactive oxygen species accumulation, mitochondrial dysfunction, and phosphatidylserine externalization in C17 fengycin B-treated F. oxysporum cells. Further data showed that C17 fengycin B induces cell apoptosis in a metacaspase-dependent manner. Importantly, we found that the expression of autophagy-related genes in the TOR signaling pathway was significantly upregulated; simultaneously, the accumulation of acidic autophagy vacuoles in F. oxysporum cell indicated that the autophagy pathway was activated during apoptosis induced by C17 fengycin B. Therefore, this study provides new insights into the antifungal mechanism of fengycin.

The overuse of chemical fungicides against fungal pathogens adversely affects soil and plant health, resulting in environmental problems and food safety. Therefore, biocontrol is considered as an environmentally friendly and cost-effective green technique in environmental protection and agricultural production. We obtained a bacterial strain N23 from a contaminated plate which showed significant inhibition to anthracnose. The strain N23 was identified as Bacillus velezensis based on 16S rRNA gene, gyrA gene, and whole-genome sequence. The bacterium N23 was able to suppress the mycelial growth of numerous plant pathogenic fungi on solid media. Tomato seeds treated with strain N23 showed significantly higher germination levels than untreated ones. Moreover, strain N23 effectively reduced the lesion area of pepper anthracnose disease in planta. The gene clusters responsible for antifungal metabolites (fengycin, surfactin, and iturin) were identified in the genome sequence of N23 based on genome mining and PCR. Furthermore, methanol extracts of the bacterial culture caused significant inhibition in growth of the fungal Colletotrichum sp. and Botrytis cinerea. These findings suggested that B. velezensis N23 could be a potential biocontrol agent in agricultural production and a source of antimicrobial compounds for further exploitation.

Alternaria spp. and its toxins are the main contaminants in processing tomato. Based on our earlier research, the current study looked into the anti-fungal capacity of crude lipopeptides from B. amyloliquefaciens XJ-BV2007 against A. alternata. We found that the crude lipopeptides significantly inhibited A. alternata growth and reduced tomato black spot disease incidence. SEM analysis found that the crude lipopeptides could change the morphology of mycelium and spores of A. alternata. Four main Alternaria toxins were detected using UPLC-MS/MS, and the findings demonstrated that the crude lipopeptides could lessen the accumulation of Alternaria toxins in vivo and in vitro. Meanwhile, under the stress of crude lipopeptides, the expression of critical biosynthetic genes responsible for TeA, AOH, and AME was substantially down-regulated. The inhibitory mechanism of the crude lipopeptides was demonstrated to be the disruption of the mycelial structure of A. alternata, as well as the integrity and permeability of the membrane of A. alternata sporocytes. Taken together, crude lipopeptides extracted from B. amyloliquefaciens XJ-BV2007 are an effective biological agent for controlling tomato black spot disease and Alternaria toxins contamination.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) poses a major challenge to vaccines and antiviral therapeutics due to their extensive evasion of immunity. Aiming to develop potent and broad-spectrum anticoronavirus inhibitors, we generated A1-(GGGGS)7-HR2m (A1L35HR2m) by introducing an angiotensin-converting enzyme 2 (ACE2)-derived peptide A1 to the N terminus of the viral HR2-derived peptide HR2m through a long flexible linker, which showed significantly improved antiviral activity. Further cholesterol (Chol) modification at the C terminus of A1L35HR2m greatly enhanced the inhibitory activities against SARS-CoV-2, SARS-CoV-2 VOCs, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses, with IC50 values ranging from 0.16 to 5.53 nM. A1L35HR2m-Chol also potently inhibits spike-protein-mediated cell-cell fusion and the replication of authentic Omicron BA.2.12.1, BA.5, and EG.5.1. Importantly, A1L35HR2m-Chol distributed widely in respiratory tract tissue and had a long half-life (>10 h) in vivo. Intranasal administration of A1L35HR2m-Chol to K18-hACE2 transgenic mice potently inhibited Omicron BA.5 and EG.5.1 infection both prophylactically and therapeutically.