Search Results (24)

To enhance the L-threonine synthesis level in Escherichia coli, this study constructed screening markers rich in L-threonine rare codons. By replacing all the threonine codons in the protein sequences with a high proportion of threonine with L-threonine rare codons and linking them to the fluorescent proteins with the same replacement, high-throughput screening of L-threonine production mutant strains was achieved. To address the metabolic imbalance caused by overexpression of a single enzyme, an artificial multi-enzyme complex system was constructed based on the principle of cellulosome self-assembly. By co-locating ThrC-DocA and ThrB-CohA, the substrate transfer path was shortened, achieving a 31.7% increase in L-threonine production. Furthermore, combined with multi-copy chromosomal integration technology via CRISPR-associated transposase (MUCICAT) technology, the thrC-docA-thrB-cohA gene cluster was integrated into the genome of the high-yield strains obtained through screening, eliminating the plasmid-dependent metabolic burden and significantly enhancing genetic stability. The modular assembly of metabolic pathways by using cellulosome elements provides a new paradigm for the optimization of complex pathways and lays a theoretical and technical foundation for the efficient production of L-threonine. Full article

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells during which parts of the cytoplasm are sequestered and subsequently delivered into lysosomes for degradation by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating the regulation and mechanism of autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. In addition, starvation-induced autophagy has not been characterized in detail using these reporters. Here, we describe the development of two endogenous autophagy reporters, gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. We demonstrate that these single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we reveal that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event because the vast majority of the detected fluorescent signals are autolysosome-specific, (ii) starvation triggers autophagy only after a measurable lag phase rather than immediately, and (iii) the regulation of starvation-induced autophagy depends on the actual life stage, and prevents excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to further dissect the physiological and pathological roles of autophagy in vivo. Full article

α-Linolenic acid (ALA) is an important essential omega-3 (ω-3) polyunsaturated fatty acid for the maintenance of human health. Although ALA has traditionally been obtained from plant sources, microbial fermentation has emerged as a promising alternative for its sustainable and cost-effective production. However, most of the present approaches rely on genetically modified organisms, which present regulatory and consumer-acceptance concerns. In this study, we aimed to develop a high-ALA-producing strain of Aspergillus oryzae, a Generally Recognized As Safe (GRAS) microorganism widely used in food production in Japan, through self-cloning, a form of genetic engineering that utilizes only the host’s own DNA. To achieve this, an endogenous ω-3 desaturase gene (fad3), which catalyzes the conversion of linoleic acid to ALA, was identified via BLASTP analysis. Subsequently, a multicopy A. oryzae strain (Aofad3-MC) overexpressing fad3 was constructed. This strain increased ALA production, with ALA comprising 30.7% of the total lipids. Furthermore, k-mer analysis confirmed the absence of foreign vector sequences, verifying that Aofad3-MC was constructed through self-cloning. In addition to the identification of the A. oryzae ω-3 desaturase gene, this study provides a microbial platform for the sustainable production of ALA, with potential applications across the food, feed, and related industries. Full article

Alkaline pectate lyases hold significant promise for various industrial applications, including the degumming processes in papermaking and textiles. In this study, a novel pectinase, PelA, derived from a strain of Paenibacillus borealis, was characterized both at the molecular level and through enzymatic analysis. This enzyme represents a distinct cluster diverging from the well-characterized Bacillus pectinases and exhibits molecular activity under alkaline conditions, with an optimal pH of 9.5. It can be classified as an endo-(1,4)-pectate lyase, capable of cleaving the α-1,4 glycosidic bonds of polygalacturonic acid via a trans-elimination mechanism. Notably, the addition of the metal ion Ca²⁺ did not enhance enzyme activity. To achieve high-level secretory expression and improve its economic viability for bioapplications, the gene copy number of pelA in the host genome was increased by constructing tandem pelA gene expression cassettes. Following optimization of cultivation conditions and monitoring of cell growth, the recombinant strain harboring the multi-copy pelA gene attained an expression level of 7520 U/mL in a bioreactor. This study successfully achieved high-level secretory expression of an alkaline pectinase, thereby enhancing its potential for industrial bioapplications and providing a reference for future research on the heterologous expression of target genes. Full article

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease, and one of the most important parasitic diseases worldwide. The first genome of T. cruzi was sequenced in 2005, and its complexity made assembly and annotation challenging. Nowadays, new sequencing methods have improved some strains’ genome sequence and annotation, revealing this parasite’s extensive genetic diversity and complexity. In this review, we examine the genetic diversity, the genomic structure, and the principal multi-gene families involved in the pathogenicity of T. cruzi. The T. cruzi genome sequence is divided into two compartments: the core (conserved) and the disruptive (variable in length and multicopy gene families among strains). The disruptive region has also been described as genome plasticity and plays a key role in the parasite survival and infection process. This region comprises several multi-gene families, including trans-sialidases, mucins, and mucin-associated surface proteins (MASPs). Trans-sialidases are the most prevalent genes in the genome with a key role in the infection process, while mucins and MASPs are also significant glycosylated proteins expressed on the parasite surface, essential for its biological functions, as host–parasite interaction, host cell invasion or protection against the host immune system, in both insect and mammalian stages. Collectively, in this review, some of the most recent advances in the structure and composition of the T. cruzi genome are reviewed. Full article

The thymidylate kinase (tmk) gene is indispensable for the proliferation and survival of phytoplasma. To reveal the molecular variation and phylogeny of the tmk genes of Candidatus phytoplasma ziziphi, in this study, the tmk genes of 50 phytoplasma strains infecting different resistant and susceptible jujube cultivars from different regions in China were amplified and analyzed. Two sequence types, tmk-x and tmk-y, were identified using clone-based sequencing. The JWB phytoplasma strains were classified into three types, type-X, type-Y, and type-XY, based on the sequencing chromatograms of the tmk genes. The type-X and type-Y strains contained only tmk-x and tmk-y genes, respectively. The type-XY strain contained both tmk-x and tmk-y genes. The type-X, type-Y, and type-XY strains comprised 42%, 12%, and 46% of all the strains, respectively. The type-X and type-XY strains were identified in both susceptible and resistant jujube cultivars, while type-Y strain was only identified in susceptible cultivars. Phylogenetic analysis indicated that the tmk genes of the phytoplasmas were divided into two categories: phylo-S and phylo-M. The phylo-S tmk gene was single-copied in the genome, with an evolutionary pattern similar to the 16S rRNA gene; the phylo-M tmk gene was multi-copied, related to PMU-mediated within-genome transposition and between-genome transfer. Furthermore, the phylogenetic tree suggested that the tmk genes shuttled between the genomes of the Paulownia witches’ broom phytoplasma and JWB phytoplasma. These findings provide insights into the evolutionary and adaptive mechanisms of phytoplasmas. Full article

(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0–8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings. Full article

The accurate diagnosis and identification of Leishmania species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for Leishmania species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using L. donovani, L. major, and L. tropica standard strain DNA. The assay was applied on 93 clinical samples with confirmed Leishmania infection, including VL (n = 30), PKDL (n = 50), and CL (n = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for L. major and up to 0.1 pg for L. donovani and L. tropica. The detection limit for the 7SL-RNA HRM-PCR was 1 pg for L. major and 10 pg for L. donovani and L. tropica. The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL (p = 0.007), PKDL (p = 0.0002), and CL (p = 0.03) samples. The ITS-1 HRM-PCR assay could identify Leishmania spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions. Full article

Isoflavones are predominantly found in legumes and play roles in plant defense and prevention of estrogen-related diseases. Genistein is an important isoflavone backbone with various biological activities. In this paper, we describe how a cell factory that can de novo synthesize genistein was constructed in Saccharomyces cerevisiae. Different combinations of isoflavone synthase, cytochrome P450 reductase, and 2-hydroxyisoflavone dehydratase were tested, followed by pathway multicopy integration, to stably de novo synthesize genistein. The catalytic activity of isoflavone synthase was enhanced by heme supply and an increased intracellular NADPH/NADP⁺ ratio. Redistribution of the malonyl-CoA flow and balance of metabolic fluxes were achieved by adjusting the fatty acid synthesis pathway, yielding 23.33 mg/L genistein. Finally, isoflavone glycosyltransferases were introduced into S. cerevisiae, and the optimized strain produced 15.80 mg/L of genistin or 10.03 mg/L of genistein-8-C-glucoside. This is the first de novo synthesis of genistein-8-C-glucoside in S. cerevisiae, which is advantageous for the green industrial production of isoflavone compounds. Full article

E2-Spy (abbreviated as ES) plays a vital role as a component in the Bacterial-Like Particles (BLPs) vaccine against classical swine fever virus (CSFV). This vaccine demonstrates remarkable immunoprotection, highlighting the importance of augmenting ES production in the development of CSFV subunit vaccines. In this study, a Pichia pastoris strain capable of high-yield secretory production of ES was developed through signal peptide engineering, gene dosage optimization and co-expression of molecular chaperones. Initially, a hybrid signal peptide cSP3 was engineered, leading to a 3.38-fold increase in ES production when compared to the control strain 1-α-ES. Subsequently, cSP3 was evaluated for its expression efficiency alongside different commonly used signal peptides under multicopy conditions. SDS-PAGE analysis revealed that 2-αd14-ES exhibited the highest ES production, displaying a 4.38-fold increase in comparison to 1-α-ES. Afterwards, SSA1, YDJ1, BIP, LHS1, and their combinations were integrated into 2-αd14-ES, resulting in a 1.92-fold rise in ES production compared to 2-αd14-ES (equivalent to a 6.18-fold increase compared to 1-α-ES). The final yield of ES was evaluated as 168.3 mg/L through comparison with serially diluted BSA protein bands. Full article

In this study, delta-12 desaturase was overexpressed in Yarrowia lipolytica using the single-copy integrative vector pINA1312 and multicopy integrative vector pINA1292, resulting in the engineered yeast strains 1312-12 and 1292-12, respectively. The content of intracellular linoleic acid (LA) in the 1292-12 strain was much higher than in the 1312-12 strain and the control group. One interesting finding was that the 1292-12 strain showed obvious changes in surface morphology. The 1292-12 colonies were much smaller and smoother, whereas their single cells became much larger compared to the control strain. In addition, the dry cell weight (DCW) of the 1292-12 strain was obviously increased from 8.5 to 12.7 g/L, but the viable cell number sharply decreased from 10⁷ to 10⁵/mL. These results indicated that increased LA content in Yarrowia lipolytica could induce morphological changes or even oxidative stress-dependent cell death. The reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated in the 1292-12 strain, while the antioxidant activities of intracellular catalase (CAT) and superoxide dismutase (SOD) were significantly decreased by 27.6 and 32.0%, respectively. Furthermore, it was also revealed that these issues could be ameliorated by the exogenous supplementation of vitamin C, fish and colza oil. Full article

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future. Full article

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate Mut^S strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii Mut^S strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes. Full article

Toxoplasmosis is a parasitic zoonosis of veterinary importance, with implications for public health. Toxoplasma gondii infection causes abortion or congenital disease in small ruminants. Moreover, the consumption of infected meat, cured meat products, or unpasteurized milk and dairy products can facilitate zoonotic transmission. Serological studies conducted in various European countries have shown the high seroprevalence of specific anti-T. gondii antibodies in sheep and goats related to the presence of oocysts in the environment, as well as climatic conditions. This article presents the current status of the detection possibilities for T. gondii infection in small ruminants and their milk. Serological testing is considered the most practical method for diagnosing toxoplasmosis; therefore, many studies have shown that recombinant antigens as single proteins, mixtures of various antigens, or chimeric proteins can be successfully used as an alternative to Toxoplasma lysate antigens (TLA). Several assays based on DNA amplification have been developed as alternative diagnostic methods, which are especially useful when serodiagnosis is not possible, e.g., the detection of intrauterine T. gondii infection when the fetus is not immunocompetent. These techniques employ multicopy sequences highly conserved among different strains of T. gondii in conventional, nested, competitive, and quantitative reverse transcriptase-PCR. Full article

Pepsinogen A (PGA) plays an important role in the treatment of human gastrointestinal diseases. At present, PGA is mainly extracted from pig stomach, so its source is very limited and its price is very expensive. Production of PGA by microbial fermentation using an engineered strain with high PGA yield would be an ideal solution. This paper presents a new system for the high-level expression of PGA from Homo sapiens (hPGA) in Aspergillus niger. The hPGA5 gene codon was optimized according to the codon bias of A. niger and then connected to a strong promoter and signal peptide to construct an hPGA5 expression cassette. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of hPGA in A. niger. By optimizing the copy number and integration sites of the hPGA5 gene, an engineering strain with a high yield of hPGA was constructed. After shake-flask fermentation for 7 d, the enzyme activity of recombinant hPGA reached 542.3 U/mL, which is the highest known activity. This lays a foundation for the production of hPGA by microbial fermentation. Full article