Cell envelope proteinases have long played a pivotal role in dairy science. However, as demand for alternative food sources grows, their application in plant-based food matrices is scarcely investigated. A deeper physiological and technological understanding of these enzymes requires efficient and effective tools tailored to this emerging sector. To date, plasmid-based recombination in Lactococcus spp. remains the most widely used and effective method. In this study, we engineered S. thermophilus LMD-9 as a host for heterologous expression of protease from Lc. cremoris. S. thermophilus LMD-9 offers several advantages, including its GRAS status, efficient chromosomal gene integration via natural competence, and native machinery for functional protease production, making it a highly versatile host. Previous attempts to employ this strain yielded inactive protease due to unresolved bottlenecks; here, we characterize and overcome those challenges to establish LMD-9 as a robust system for protease expression. Exploration of potential bottlenecks highlighted the availability of intrinsic peptidyl-prolyl cis/trans isomerase (PPIase; prtM/prsA) as a key factor influencing successful enzyme expression. Three recombinant strains with genotypes: LMD-9 ΔprtS::ermR, LMD-9 ΔprtS::ermR-ΩprtP, and LMD-9 ΔprtS::ermR-Ω (prtM-prtP), were generated to test the role of prtP-associated PPIase. Results demonstrated that inclusion of PrtP-specific PPIase from L. cremoris markedly enhances protease activity. In its absence, although partially compensated by the pleiotropic PPIase in S. thermophilus, we observed slower growth and reduced proteolytic activity. These findings establish S. thermophilus LMD-9 as a robust chassis and alternative host for heterologous expression of cell envelope proteinases. To our knowledge, this is the first work to heterologously express active CEP enzyme in this host, and it highlights the role of PPIase availability as a key determinant of successful enzyme expression. This then provides a suitable and robust host for studying the application of CEPs in both dairy and emerging plant-based food applications.

Biopesticides constitute a category of natural products and organisms effective against pests. Given their advantages, such as easier clearance and degradation compared to chemical pesticides, along with their lack of detrimental effects on ecosystem health, they represent a promising alternative for sustainable pest management and crop protection. The development of natural biopesticides to substitute synthetic pesticides is therefore of paramount importance for securing food safety and advancing agricultural production. Monoterpenes, primarily present in plant volatile oils, are characterized by a strong aroma and a range of biological activities-including insecticidal, antimicrobial, antiviral, anti-inflammatory, antioxidant, antitumor, and anticancer effects. This profile renders them a promising platform for the research and development of new, more effective pesticides. This article reviews the biosynthetic pathways of monoterpenes and contemporary engineering strategies for their microbial synthesis. Additionally, an evolutionary analysis database for monoterpene synthases (MTPS) has been established. Finally, the biological activities of monoterpenes are summarized and analyzed, with particular emphasis on their potential and advantages as biopesticides, thereby providing direction for the future development of monoterpene-based biopesticides.

Halophiles attract increasing attention to serve as sustainable industrial hosts for prolonged continuous processes or in open-vat fermentations owing to the reduced risk of contamination enabled by high salt concentration in the medium. Despite growing interest, their application in large-scale manufacturing remains limited partly due to bioprocessing challenges. A robust host that performs consistently well across scales should withstand variations in oxygen availability since local hypoxic regions often manifest in large-scale tanks even under strict operational control. In this work, we modified Halomonas sp. to achieve robust growth profiles in micro-aerobic environments by gradually exposing a continuous culture to stress induced by reduced oxygen availability using adaptive laboratory evolution. Nominal contamination was observed at 8.5% salt concentration even during non-aseptic operation, offering a suitable environment for prolonged continuous cultivation. Following adaptive laboratory evolution, Halomonas sp. achieved comparable growth at low-to-moderate (0.5%-3.0%) and moderate-to-high (3.0–5.0%) dissolved oxygen availability (OD600 = 4.46 ± 0.29 & µ = 0.40 h− 1 and 4.96 ± 0.81 & µ = 0.35 h− 1, respectively). Adaptive evolution improved the growth robustness of Halomonas sp. by 62% and its PHB production yield by 21%, while reducing ectoine production by 79% indicting improved metabolic perception of oxygen availability and reduced stress response. Mutations in cellular transport mechanisms and enzymatic pathways enabled this response as indicated by whole genome sequencing. The proposed framework is an effective strategy to reduce the oxygen sensitivity of microbial strains in general to improve their physiological properties, and to genetically design populations suitable for large scale bioprocess operations.

The utilisation of microbes for the production of enzymes and pharmaceutical proteins is an important step towards a sustainable future. However, the energy requirement for agitation, aeration and cooling during industrial fermentation processes is substantial. To address this challenge, we have previously developed a 'breathing' polytetrafluoroethylene fermenter vessel that allows effective gas exchange with ambient air and, thus, does not require sparger aeration. The present study aimed to explore the potential application of the breathing vessel for enzyme production by the Gram-positive bacterial cell factory Bacillus subtilis. Here, we compared production of the secreted α-amylase AmyQ by the genome-reduced multiple protease-deficient B. subtilis strain IIG-Bs-27-31 during parallel culturing in breathing vessels and shake flasks. Enzyme yields and the cellular and extracellular proteome compositions were assessed. We observed comparable growth characteristics and AmyQ yields per liter in both culture systems. However, proteome analyses indicated statistically significant differences (p < 0.05, log2fold change>|0.5|) in the utilization of carbon sources and stress responses between cells grown in the breathing vessels versus shake flasks. In particular, bacteria in the breathing vessel presented activation of the Sigma B-dependent general stress response, presumably to sustain bacterial growth and viability. In contrast, bacteria grown in shake flask presented activated cell envelope stress pathways and typical sheer stress symptoms. While the overall AmyQ yields per liter were similar in both fermentation systems, the total enzyme yields in the breathing fermenter were significantly higher due to the 15-fold increase in culture volume. Our findings imply that breathing fermentation vessels are suitable for B. subtilis enzyme production and, upon further scale-up, they may represent sustainable and cost-effective alternatives to traditional fermentation systems for microbial cell factories.

Menaquinone-7 (MK-7), a highly bioactive form of vitamin K₂ with a long half-life, plays pivotal roles in preventing osteoporosis and cardiovascular diseases. A metabolically balanced MK-7-producing strain, Bacillus subtilis BS016, has been engineered. However, its biosynthetic efficiency remains hindered by bottlenecks, such as low isoprenoid side-chain elongation efficiency. To address this limitation, a thermophilic farnesyl diphosphate (FPP) synthase from Geobacillus stearothermophilus, characterized by high activity and stability, was identified via database mining. This enzyme was modularly assembled with the endogenous hepS-menG-hepT operon in B. subtilis BS016, driven by the strong promoter Phbs. A synergistic expression cassette was constructed to achieve spatial co-localization of FPP synthesis with downstream isoprenoid side-chain elongation. The resulting engineered strain BS018 achieved a flask yield of 91.1 mg/L, representing an 11% increase, and maintained a stable titer of 87.9 mg/L in a 5-L fermenter. Further optimisation of fermentation conditions (liquid loading, 50 mL/500 mL; initial pH, 7.0; inoculum dose, 8%) ultimately elevated a flask yield to 109.6 mg/L. This study demonstrates that the directed screening of high activity heterologous enzymes, combined with the spatial co-assembly of endogenous pathways, can effectively reconstruct and enhance metabolic flux. This integrated strategy, combining directed enzyme screening with spatial pathway assembly, provides a generalizable framework for constructing efficient cell factories not only for vitamin K₂ but also for other high-value terpenoids.

BackgroundCyanobacteria and their bioactive components represent a promising potential for the green synthesis of nanoparticles. This work investigates the physicochemical characteristics and biological activities of silver nanoparticles (PC-AgNPs) synthesized using phycocyanin, a photosynthetic pigment extracted from the cyanobacterium Arthrospira platensis Pc-005.ResultsThe phycocyanin-derived AgNPs were characterized using UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Energy Dispersive X-Ray Spectroscopy (EDX), and Dynamic light scattering (DLS). Physicochemical analysis confirmed the formation of PC-AgNPs, and revealed their nanoscale size, crystalline nature and purity. PC-AgNPs demonstrated a UV-Vis absorption peak at 410 nm. TEM analysis revealed that phycocyanin-derived AgNPs were spherical in shape with an average diameter of 20.0 ± 4.2 nm. XRD and EDX analyses confirmed the crystallinity and purity of PC-AgNPs. FTIR analysis revealed the involvement of phycocyanin functional groups in AgNPs formation. The results demonstrated that PC-AgNPs exhibited strong antimicrobial activity against opportunistic pathogenic bacteria Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, as well as fungi Aspergillus flavus, Penicillium aurantioviolaceum, and Trichoderma viride. The findings indicated that significantly higher concentration of PC-AgNPs was needed to achieve antifungal activity compared to antibacterial activity. The PC-AgNPs also affected the activity of crucial antioxidant enzymes, such as superoxide dismutase and catalase, which play key role in the bacterial defense against oxidative stress induced by AgNPs. Moreover, PC-AgNPs demonstrated significant concentration-dependent anticancer activity against A549 adenocarcinoma cells. They also inhibited key angiogenic factors, including VEGF-A and MMP-2, as well as inflammation-related markers such as TNF-α and COX-2.ConclusionThus, this study provides a comprehensive physicochemical and biological characterization of silver nanoparticles derived from the phycocyanin of A. platensis. The PC-AgNPs exhibit a broad spectrum of biological activities, underscoring their promising potential for innovative applications in biomedical and biotechnological fields.Graphical

BackgroundThe development of antimicrobial treatments as alternatives to antibiotics to combat antimicrobial-resistant (AMR) bacteria is a global priority. Antimicrobial peptides and proteins such as Host Defense Peptides (HDPs) and endolysins are one of the alternatives that are being explored. HDPs are small, cationic, and amphiphilic antimicrobial peptides derived from the innate immune system exhibiting a broad-spectrum antimicrobial activity. On the other hand, endolysins are enzymes produced by bacteriophages to hydrolyze the bacterial peptidoglycan layer, offering more specific antimicrobial activity than HDPs. While short peptides can be chemically synthesized, this approach presents several limitations, and recombinant production is also being used. Escherichia coli is the most used bacterial expression system for protein production. Alternative systems based on Generally Recognized as Safe (GRAS) microorganisms such as Lactic Acid Bacteria (LAB) have also been employed. However, so far, no comparative studies have evaluated the production and activity of antimicrobial proteins expressed in E. coli versus LAB and this study aims to address that gap.ResultsTo evaluate potential differences in the production of antimicrobial proteins using E. coli and two LAB (Lactococcus lactis and Lactiplantibacillus plantarum) hosts, various proteins were evaluated. These included two HDPs fused to a GFP, two multidomain HDP-based proteins and one endolysin. The results revealed a clear influence of the expression system on the quality of HDP-based protein, including both GFP fusions and multidomain constructs. Protein yield was higher in E. coli and all HDP-based proteins exhibited higher antimicrobial activity when expressed in E. coli compared to L. lactis and L. plantarum. In contrast, endolysin activity was comparable when produced in E. coli and L. lactis.ConclusionsThese results demonstrate that the choice of bacterial expression host significantly affects not only the yield but, more importantly, the antimicrobial activity of HDP-based proteins. For these proteins, the antimicrobial activity was consistently higher when produced in E. coli. In contrast, endolysins exhibited similar characteristics regardless of whether they were expressed in E. coli or in L. lactis.