Expression Host Research Articles

Identification and Characterization of a Fungal Monoterpene Synthase Responsible for the Biosynthesis of Geraniol.

Geraniol, an acyclic monoterpenoid of substantial value extracted from the essential oils of various aromatic plants, holds significant commercial and industrial importance in the realms of food, cosmetics, medicine, and bioenergy. Geraniol synthase, which is responsible for geraniol production, has been identified in only several plant species to date. Here, we present the first cloning and characterization of a geraniol synthase (PgfTPS) from Penicillium griseofulvum. This enzyme demonstrates pronounced specificity in catalyzing the conversion of geranyl diphosphate into geraniol. Moreover, through protein modeling and site-directed mutagenesis, we have identified key active-site residues crucial for the catalytic function of PgfTPS. Finally, we utilized engineered Saccharomyces cerevisiae as a host for PgfTPS expression to facilitate geraniol production. Our findings not only advance the development of efficient biocatalysts for geraniol generation but also establish a fundamental basis for further exploration into fungal monoterpene biosynthesis.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

ABSTRACTChlamydomonas incerta, a genetically close relative of the model green algaChlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship betweenC. incertaandC. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly withC. reinhardtii,C. incertaexhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed thatC. incertaproduced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared toC. reinhardtii. For xylanase expression and secretion, bothC. incertaandC. reinhardtiishowed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally,C. incertashowed comparable PHL7 activity levels toC. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids betweenC. incertaandC. reinhardtiias a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasizeC. incerta’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.GRAPHICAL ABSTRACT

Characterization of a High-Affinity Copper Transporter CTR1a in the White-Nose Syndrome Causing Fungal Pathogen Pseudogymnoascus destructans.

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a can efficiently traffic Cu ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu homeostasis pathways in P. destructans.

Identification and analysis of the key genes for Escherichia coli heterologous protein expression by transcriptomic profiling.

Escherichia coli is a frequently used host for heterologous protein expression, but its expression efficiency is hindered by several limitations, such as formation of inclusion bodies and proteolytic degradation. In this study, we employed high-density fermentation of heterologous protein production in a 5-L bioreactor, resulting in a yield 2.25 times higher than that of the control group. Transcriptional analysis was conducted at three time points after induction for 0h, 4h, and 12h, revealing 420, 301, and 570 upregulated differentially expressed genes, as well as 424, 202, and 525 downregulated genes, respectively. By conducting enrichment analysis, we constructed strains that relieved without iron limitation, exhibiting a 36% increase in biomass and a 32% increase in protein expression. Furthermore, no overflow metabolism of acetic acid was detected during the protein expression process when utilizing chemostat culture, which indicated that the utilization efficiency of glucose was significantly enhanced without iron limitation. This study presents a novel approach to better comprehend the mechanism of high-yield production of heterologous proteins in Escherichia coli.

Comparative Analysis of the Codon Usage Pattern in the Chloroplast Genomes of Gnetales Species

Codon usage bias refers to the preferential use of synonymous codons, a widespread phenomenon found in bacteria, plants, and animals. Codon bias varies among species, families, and groups within kingdoms and between genes within an organism. Codon usage bias (CUB) analysis sheds light on the evolutionary dynamics of various species and optimizes targeted gene expression in heterologous host plants. As a significant order of gymnosperms, species within Gnetales possess extremely high ecological and pharmaceutical values. However, comprehensive analyses of CUB within the chloroplast genomes of Gnetales species remain unexplored. A systematic analysis was conducted to elucidate the codon usage patterns in 13 diverse Gnetales species based on the chloroplast genomes. Our results revealed that chloroplast coding sequences (cp CDSs) in 13 Gnetales species display a marked preference for AT bases and A/T-ending codons. A total of 20 predominantly high-frequency codons and between 2 and 7 optimal codons were identified across these species. The findings from the ENC-plot, PR2-plot, and neutrality analyses suggested that both mutation pressure and natural selection exert influence on the codon bias in these 13 Gnetales species, with natural selection emerging as the predominant influence. Correspondence analysis (COA) demonstrated variation in the codon usage patterns among the Gnetales species and indicated mutation pressure is another factor that could impact CUB. Additionally, our research identified a positive correlation between the measure of idiosyncratic codon usage level of conservatism (MILC) and synonymous codon usage order (SCUO) values, indicative of CUB’s potential influence on gene expression. The comparative analysis concerning codon usage frequencies among the 13 Gnetales species and 4 model organisms revealed that Saccharomyces cerevisiae and Nicotiana tabacum were the optimal exogenous expression hosts. Furthermore, the cluster and phylogenetic analyses illustrated distinct patterns of differentiation, implying that codons, even with weak or neutral preferences, could affect the evolutionary trajectories of these species. Our results reveal the characteristics of codon usage patterns and contribute to an enhanced comprehension of evolutionary mechanisms in Gnetales species.

More than just the gene: investigating expression using a non-native plasmid and host and its impact on resistance conferred by β-lactamase OXA-58 isolated from a hospital wastewater microbiome.

With the escalation of hospital-acquired infections by multidrug resistant bacteria, understanding antibiotic resistance is of paramount importance. This study focuses on the β-lactamase gene, blaOXA-58, an important resistance determinant identified in a patient-facing hospital wastewater system. This study aimed to characterize the behaviour of the OXA-58 enzyme when expressed using a non-native plasmid and expression host. blaOXA-58 was cloned using a pET28a(+)/Escherichia coli BL21(DE3) expression system. Nitrocefin hydrolysis and antimicrobial susceptibility of OXA-58-producing cells were assessed against penicillin G, ampicillin, meropenem, and amoxicillin. blaOXA-58 conferred resistance to amoxicillin, penicillin G, and ampicillin, but not to meropenem. This was unexpected given OXA-58's annotation as a carbapenemase. The presence of meropenem also reduced nitrocefin hydrolysis, suggesting it acts as a competitive inhibitor of the OXA-58 enzyme. This study elucidates the phenotypic resistance conferred by an antimicrobial resistance gene (ARG) obtained from a clinically relevant setting and reveals that successful functional expression of ARGs is multifaceted. This study challenges the reliability of predicting antimicrobial resistance based solely on gene sequence alone, and serves as a reminder of the intricate interplay between genetics and structural factors in understanding resistance profiles across different host environments.

N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase

Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in kcat. These substrates with a short chain of 2–4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the −1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.

GenRCA: a user-friendly rare codon analysis tool for comprehensive evaluation of codon usage preferences based on coding sequences in genomes

BackgroundThe study of codon usage bias is important for understanding gene expression, evolution and gene design, providing critical insights into the molecular processes that govern the function and regulation of genes. Codon Usage Bias (CUB) indices are valuable metrics for understanding codon usage patterns across different organisms without extensive experiments. Considering that there is no one-fits-all index for all species, a comprehensive platform supporting the calculation and analysis of multiple CUB indices for codon optimization is greatly needed.ResultsHere, we release GenRCA, an updated version of our previous Rare Codon Analysis Tool, as a free and user-friendly website for all-inclusive evaluation of codon usage preferences of coding sequences. In this study, we manually reviewed and implemented up to 31 codon preference indices, with 65 expression host organisms covered and batch processing of multiple gene sequences supported, aiming to improve the user experience and provide more comprehensive and efficient analysis.ConclusionsOur website fills a gap in the availability of comprehensive tools for species-specific CUB calculations, enabling researchers to thoroughly assess the protein expression level based on a comprehensive list of 31 indices and further guide the codon optimization.

Characterization of Escherichia coli as a Recombinant Protein Expression Host and its Optimization

Engineered Escherichia coli serves as an efficient platform for the production of a diverse array of recombinant proteins, thereby addressing the pharmaceutical industry's demand for high-quality biological agents. Nevertheless, challenges related to protein folding and the complexities involved in product purification must be acknowledged. This review delineates the advantages and disadvantages of utilizing E. coli as an expression host and further investigates various strategies aimed at optimizing and enhancing the E. coli expression system to elevate the yield, purity, and biological activity of recombinant proteins, ultimately supporting advancements in biopharmaceuticals and other relevant fields.

Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440.

p-Coumaric acid (p-CA) is a valuable compound with applications in food additives, cosmetics, and pharmaceuticals. However, traditional production methods are often inefficient and unsustainable. This study focuses on enhancing p-CA production efficiency through the heterologous expression of tyrosine ammonia-lyase (TAL) from Rhodobacter sphaeroides in Pseudomonas putida KT2440. TAL catalyzes the conversion of L-tyrosine into p-CA and ammonia. We engineered P. putida KT2440 to express TAL in a fed-batch fermentation system. Our results demonstrate the following: (i) successful integration of the TAL gene into P. putida KT2440 and (ii) efficient bioconversion of L-tyrosine into p-CA (1381 mg/L) by implementing a pH shift from 7.0 to 8.5 during fed-batch fermentation. This approach highlights the viability of P. putida KT2440 as a host for TAL expression and the successful coupling of fermentation with the pH-shift-mediated bioconversion of L-tyrosine. Our findings underscore the potential of genetically modified P. putida for sustainable p-CA production and encourage further research to optimize bioconversion steps and fermentation conditions.

Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography.

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure-property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

Heterologous expression of GH11 xylanase from Myceliophthora heterothallica F.2.1.4 in Pichiapastoris

This research is centered on improving a xylanase enzyme derived from Myceliophthora heterothallica by expressing it in Pichia pastoris to enhance biomass hydrolysis. Lignocellulosic biomass holds significant potential for renewable applications in biofuels, chemicals, and pharmaceuticals. The study explores the impact of the choice of expression host on enzyme properties, specifically addressing challenges in thermostability by utilizing P. pastoris and leveraging its glycosylation capabilities. Previously expressed in Escherichia coli, the xylanase exhibited in this work cooperative kinetics, pH stability, and resistance to phenolic compounds. Gene integration and expression in P. pastoris were verified through PCR and activity assays. After 120 h of induction, an enzymatic activity of 48.8 U mL−1 was obtained. Subsequent characterization revealed improved specific activity, substrate affinity, and optimal temperature compared to the enzyme expressed in E. coli. The enzyme exhibited excellent pH and temperature stability for industrial applications, maintaining over 90% of its activity within a pH range of 5.0–10.0 and remaining stable even after 90 min of incubation at 55 °C. It also demonstrated resistance to metal ions and responsiveness to phenolic compounds. These findings underscore the versatility of the recombinant xylanase from M. heterothallica expressed in P. pastoris, highlighting its potential as a valuable resource for biomass conversion. The study emphasizes the pivotal role of host choice in optimizing enzyme characteristics for industrial applications, underscoring the importance of employing heterologous expression systems, as demonstrated in this investigation.

Advancements in the Heterologous Expression of Sucrose Phosphorylase and Its Molecular Modification for the Synthesis of Glycosylated Products.

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

Characterization of a High-Affinity Copper Transporter in the White-Nose Syndrome Causing Fungal Pathogen Pseudogymnoascus destructans.

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a localizes to the cell surface plasma membrane and can efficiently traffic Cu-ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu-homeostasis pathways in P. destructans.

Expression, Purification, and Evaluation of Antibody Responses and Antibody-Immunogen Complex Simulation of a Designed Multi-Epitope Vaccine against SARS-COV-2.

The spread of the COVID-19 disease is the result of an infection caused by the SARS-CoV2 virus. Four crucial proteins, spike [S], membrane [M], nucleocapsid [N], and envelope [E] in coronaviruses have been considered to a large extent. This research aimed to express the recombinant protein of a multiepitope immunogen construct and evaluate the immunogenicity of the multiepitope vaccine that was previously designed as a candidate immunogenic against SARS-Cov-2. Plasmid pET26b was transferred to the expression host E. coli BL21 [DE3] and the recombinant protein was expressed with IPTG induction. The recombinant protein was purified by Ni-NTA column affinity chromatography, and western blotting was used to confirm it. Finally, mice were immunized with recombinant protein in three doses. Then, the interaction of the 3D structure of the vaccine with the human neutralizing antibodies3D structures [7BWJ and 7K8N] antibody was evaluated by docking and molecular dynamics simulation. The optimized gene had a codon compatibility index of 0.96. The expression of the recombinant protein of the SARS-Cov-2 vaccine in an E. coli host led to the production of the recombinant protein with a weight of about 70 kDa with a concentration of 0.7 mg/ml. Immunization of mice with recombinant protein of SARS-Cov-2 vaccine-induced IgG serum antibody response. Statistical analysis showed that the antibody titer in comparison with the control sample has a significant difference, and the antibody titer was acceptable up to 1/256000 dilution. The simulation of vaccine binding with human antibodies by molecular dynamics showed that Root Mean Square Deviation [RMSD], Root Mean Square Fluctuation [RMSF], Radius of Gyration, and H-bond as well as van der Waals energies and electrostatic of Molecular mechanics Poisson- Boltzmann surface area [MM/PBSA] analysis have stable interaction. This recombinant protein can probably be used as an immunogen candidate for the development of vaccines against SARS-CoV2 in future research.

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase

BackgroundThe large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.ResultsHerein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.ConclusionsResults show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

Producing recombinant proteins in Vibrio natriegens

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes.Key pointsConstruction of a highly efficient Penicillium crustosum heterologous expression hostReduction of secondary metabolite background by genetic dereplication strategyIntegration of wA site to provide an alternative host besides Aspergillus nidulansGraphical

Enhanced resistance of Lactiplantibacillus plantarum by expression of albumin.

Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing-strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113-pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild-type strain AR113-pIB184. The results demonstrated that the survival rate of AR113-pLLY01 was also significantly better than that of AR113-pIB184 after lyophilization. In addition, AR113-pLLY01 exhibited a significantly better protective effect than AR113-pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113-pLLY01 had a significant protective effect. This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry.

Green algae-produced multimer cathelicidin-RC1 disrupts membrane integrity for inhibiting bacterial growth

Cathelicidin-RC1, extracted from the bullfrog Rana catesbeiana, has been found to effectively combat gram-negative bacteria but has limited inhibitory effects on gram-positive bacteria. Additionally, the specific mechanism of cathelicidin-RC1’s interaction with bacteria remains unclear. In this research, we present the expression analysis of a chimeric peptide composed of three repetitive units of cathelicidin-RC1 (3×cathelicidin-RC1) with a hemagglutinin (HA) and a 6×His tag attached at its C-terminus in Chlamydomonas reinhardtii, leading to the production of a chimeric peptide with a molecular mass of ∼16.2 kDa. The recombinant peptide, 3×cathelicidin-RC1-HA-6×His, demonstrates a wide range of antimicrobial activity, with minimum inhibitory concentration (MIC) values of approximately 20–30 µg/ml and 10–60 µg/ml against gram-positive and negative bacteria, respectively. The minimum bactericidal concentration (MBC) values of 3×cathelicidin-RC1-HA-6×His varies from 1× to 3×MIC. Furthermore, 3×cathelicidin-RC1-HA-6×His demonstrates excellent stability, pH stability, and resistance to digestion by certain proteases. It also exhibits low cytotoxicity and negligible hemolytic activity against rat erythrocytes. Scanning electron microscopy and propidium iodide analysis indicate that 3×cathelicidin-RC1-HA-6×His disrupts bacterial cell membranes, leading to inhibition of bacterial growth. Overall, our findings demonstrate that 3×cathelicidin-RC1-HA-6×His, produced in the heterologous expression host C. reinhardtii, effectively inhibits bacterial growth and holds great promise for potential application.