Strong Promoter Research Articles

Improvement of succinate production from methane by combining rational engineering and laboratory evolution in Methylomonas sp. DH-1

Recently, methane has been considered a next-generation carbon feedstock due to its abundance and it is main component of shale gas and biogas. Methylomonas sp. DH-1 has been evaluated as a promising industrial bio-catalyst candidate. Succinate is considered one of the top building block chemicals in the agricultural, food, and pharmaceutical industries. In this study, succinate production by Methylomonas sp. DH-1 was improved by combining adaptive laboratory evolution (ALE) technology with genetic engineering in the chromosome of Methylomonas sp. DH-1, such as deletion of bypass pathway genes (succinate dehydrogenase and succinate semialdehyde dehydrogenase) or overexpression of genes related with succinate production (citrate synthase, pyruvate carboxylase and phosphoenolpyruvate carboxylase). Through ALE, the maximum consumption rate of substrate gases (methane and oxygen) and the duration maintaining high substrate gas consumption rates was enhanced compared to those of the parental strain. Based on the improved methane consumption, cell growth (OD600) increased more than twice, and the succinate titer increased by ~ 48% from 218 to 323 mg/L. To prevent unwanted succinate consumption, the succinate semialdehyde dehydrogenase gene was deleted from the genome. The first enzyme of TCA cycle (citrate synthase) was overexpressed. Pyruvate carboxylase and phosphoenolpyruvate carboxylase, which produce oxaloacetate, a substrate for citrate synthase, were also overproduced by a newly identified strong promoter. The new strong promoter was screened from RNA sequencing data. When these modifications were combined in one strain, the maximum titer (702 mg/L) was successfully improved by more than three times. This study demonstrates that successful enhancement of succinic acid production can be achieved in methanotrophs through additional genetic engineering following adaptive laboratory evolution.

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.

Autoregulation ensures vertical transmission of the linear prophage GIL01

Betatectiviruses are prophages consisting of linear extrachromosomal genomes without obvious plasmid modules. It remains unclear how betatectiviruses are maintained in low-copy numbers in host cells and how they are vertically transmitted. Phage GIL01 is a model betatectivirus that infects the mosquito pathogen Bacillus thuringiensis serovar israelensis. Previous studies identified two closely spaced promoters, P1 and P2, responsible for the expression of GIL01 genes required for prophage replication and the switch from the lysogenic to lytic cycle. Here, we report that the GIL01-encoded 58-amino acid long gp1 protein forms a large nucleoprotein complex that represses its transcription from the strong promoter P2. Notably, ectopic expression of gp1 resulted in the loss of GIL01 in exponential cultures and immunized cells against infection with GIL01, indicating that gp1 plays a repressive role in the phage cycle. This finding is consistent with mutations in gp1 committing GIL01 to the lytic cycle and we show that maintenance of this phage variant in the bacterial population is contingent on the accumulation of deletions in the P1-P2 region. The fact that gp1 is conserved across most sequenced betatectiviruses suggests that the regulatory mechanism of gp1 that controls prophage maintenance is widespread among these bacteriophages.

Combining culture optimization and synthetic biology to improve production and detection of secondary metabolites in Myxococcus xanthus: application to myxoprincomide.

Microbial secondary metabolites play crucial ecological roles in governing species interactions and contributing to their defense strategies. Their unique structures and potent bioactivities have been key in discovering antibiotics and anticancer drugs. Genome sequencing has undoubtedly revealed that myxobacteria constitute a huge reservoir of secondary metabolites as the well-known producers, actinomycetes. However, because most secondary metabolites are not produced in the laboratory context, the natural products from myxobacteria characterized to date represent only the tip of the iceberg. By combining the engineering of a dedicated Myxococcus xanthus DZ2 chassis strain with a two-step growth medium protocol, we provide a new approach called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) for the production of non-ribosomal peptides synthetases (NRPS)/polyketides synthases (PKS) secondary metabolites from myxobacteria. We further show that the 2PRIM-BOOST strategy will facilitate the screening of secondary metabolites for biological activities of medical interest. As proof of concept, using a constitutive strong promoter, the myxoprincomide from M. xanthus DZ2 has been efficiently produced and its biosynthesis has been enhanced using the 2PRIM-BOOST approach, allowing the identification of new features of myxoprincomide. This strategy should allow the chances to produce and discover new NRPS, PKS, and mixed NRPS/PKS hybrid natural metabolites that are currently considered as cryptic and are the most represented in myxobacteria.IMPORTANCEMicrobial secondary metabolites are important in species interactions and are also a prolific source of drugs. Myxobacteria are ubiquitous soil-dwelling bacteria constituting a huge reservoir of secondary metabolites. However, because most of these molecules are not produced in the laboratory context, one can estimate that only one-tenth have been characterized to date. Here, we developed a new strategy called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) that combines the engineering of a dedicated Myxococcus xanthus chassis strain together with growth medium optimization. By combining these strategies with the insertion of a constitutive promoter upstream the biosynthetic gene cluster (BGC), the production of myxoprincomide, a characterized low-produced secondary metabolite, was successfully and significantly increased. The 2PRIM-BOOST enriches the toolbox used to produce previously cryptic metabolites, unveil their ecological role, and provide new molecules of medical interest.

New Plasmid Constructs for Production of Yeast Based Oral Vaccines

In order to avoid conventional vaccines, there is an urgent need to look for new ways of vaccine generation that can cut down the cost, time, and the need for special requirements during storage and distribution. The development of oral peptide/protein-based vaccines could be an ideal alternative. Five expression plasmids are designed in this work for the production of Saccharomyces cerevisiae-based oral vaccines. Therefore, in this work genetic cassette with different organizations of the required genetic components for efficient gene expression is constructed in the pYES2 plasmid. The construction of five new plasmids for surface display of heterologous recombinant proteins in S. cerevisiae would enable easy insertion of the gene of interest into an optimized genetic cassette consisting of a strong host promoter GAL1, HA-tag for detection of recombinant enzymes, GAS1 gene with/without binding sequence coded for GAS1 cell wall protein and signal sequence for directing the protein into the secretory pathway. After the construction of the genetic cassettes, E gene coded for envelope protein of Chikungunya and Dengue virus is inserted in the genetic cassettes. Genetic models pYES2HAGAS1EgeneChikungunya, pYES2HAGAS1 Egene Dengue, pYES2GAS1 Egene ChikungunyaHA, and pYES2GAS1EGeneDengueHA are constructed with different genetic organizations to display envelope protein of Chikungunya and Dengue virus in the cell surface of S. cerevisiae. In the pYES2SignalEGeneDengueHA construction, envelope protein will be secreted by S. cerevisiae into the media. The constructed vectors are specialized to regulate the expression and surface display of heterologous recombinant proteins in Saccharomyces cerevisiae and will be suitable for large-scale oral vaccine production.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Genomic insights and functional evaluation of Lacticaseibacillus paracasei EG005: a promising probiotic with enhanced antioxidant activity.

Probiotics, such as Lacticaseibacillus paracasei EG005, are gaining attention for their health benefits, particularly in reducing oxidative stress. The goal of this study was to reinforce the antioxidant capacity of EG005, along with comprehensive genomic analysis, with a focus on assessing superoxide dismutase (SOD) activity, acid resistance and bile tolerance, and safety. EG005 was screened for SOD activity and change of SOD activity was tested under various pH conditions. Its survival rates were assessed in acidic (pH 2.5) and bile salt (0.3%) conditions and the antibiotic MIC test and hemolysis test were performed to evaluate safety. Genetic analyses including functional identification and phylogenetic tree construction were performed. The SOD overexpression system was constructed using Ptuf, Pldh1, Plhd2, and Pldh3 strong promoters. EG005 demonstrated higher SOD activity compared to Lacticaseibacillus rhamnosus GG, with optimal activity at pH 7.0. It showed significant acid and bile tolerance, with survival rates recovering to 100% after 3 h in acidic conditions. Phylogenetic analysis confirmed that EG005 is closely related to other L. paracasei strains with ANI values above 98%. Overexpression of SOD using the Ptuf promoter resulted in a two-fold increase in activity compared to the controls. Additionally, EG005 exhibited no hemolytic activity and showed antibiotic susceptibility within safe limits. Our findings highlight EG005's potential as a probiotic with robust antioxidant activity and high tolerance to gastrointestinal conditions. Its unique genetic profile and enhanced SOD activity through strong promoter support its application in probiotic therapies and functional foods. Further research should be investigated to find the in vivo effects of EG005 on gut health and oxidative stress reduction. In addition, attB and attP-based recombination, combined with CRISPR-Cas9 technologies, could offer a more stable alternative for long-term sodA gene expression in commercial and medical applications.

Overexpressing CsSABP2 enhances tolerance to Huanglongbing and citrus canker in C. sinensis.

Huanglongbing (HLB) and citrus canker, arising from Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. Citri (Xcc), respectively, have been imposing tremendous losses to the global citrus industry. Systemic acquired resistance (SAR) has been shown to be crucial for priming defense against pathogen in citrus. Salicylic acid (SA) binding protein 2 (SABP2), which is responsible for converting methyl salicylate (MeSA) to SA, is essential for full SAR establishment. Here, we characterized the functions of four citrus SABP2 genes (CsSABP2-1, CsSABP2-1V18A , CsSABP2-2 and CsSABP2-3) against HLB and citrus canker. In vitro enzymatic assay revealed that all four proteins had MeSA esterase activities, and CsSABP2-1 and CsSABP2-1V18A has the strongest activity. Their activities were inhibited by SA except for CsSABP2-1V18A. Four genes controlled by a strong promoter 35S were induced into Wanjincheng orange (Citrus sinensis Osbeck) to generate transgenic plants overexpressing CsSABP2. Overexpressing CsSABP2 increased SA and MeSA content and CsSABP2-1V18A had the strongest action on SA. Resistance evaluation demonstrated that only CsSABP2-1V18A had significantly enhanced tolerance to HLB, although all four CsSABP2s had increased tolerance to citrus canker. The data suggested the amino acid Val-18 in the active site of CsSABP2 plays a key role in protein function. Our study emphasized that balancing the levels of SA and MeSA is crucial for regulating SAR and conferring broad-spectrum resistance to HLB and citrus canker. This finding offers valuable insights for enhancing resistance through SAR engineering.

Genetic Transformation of Triticum dicoccum and Triticum aestivum with Genes of Jasmonate Biosynthesis Pathway Affects Growth and Productivity Characteristics.

The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana (AtAOS) and bread wheat (TaAOS) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana (AtOPR3) under the strong constitutive promoter (ZmUbi1), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS, alone or together with AtOPR3, negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double (AtAOS + AtOPR3) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds.

An in-silico approach to the dynamics of proliferation potential in stem cells and the study of different therapies in cases of ovarian dysfunction

A discrete mathematical model based on ordinary differential equations and the associated continuous model formed by a partial differential equation, which simulate the generational and temporal evolution of a stem cell population, are proposed. The model parameters are the maximum proliferation potential and the rates of mitosis, death events and telomerase activity. The mean proliferation potential at each point in time is suggested as an indicator of population aging. The model is applied on hematopoietic stem cells (HSCs), with different telomerase activity rates, in a range of variation of maximum proliferation potential in healthy individuals, to study the temporal evolution of aging. HSCs express telomerase, however not at levels that are sufficient for maintaining constant telomere length with aging [1,2]. Women with primary ovarian insufficiency (POI) are known to have low telomerase activity in granulosa cells and peripheral blood mononuclear cells [3]. Extrapolating this to hematopoietic stem cells, the mathematical model shows the differences in proliferation potential of the cell populations when telomerase expression is activated using sexual steroids, though the endogenous promoter or with gene therapy using exogenous, stronger promoters within the adeno-associated virus. In the first case, proliferation potential of cells from POI condition increases, but when adeno-associated viruses are used, the proliferation potential reaches the levels of healthy cell populations.

Original Research Paper

Plants inhabiting adverse growth conditions compete against stresses by the endogenous regulatory elements viz., promoters and terminators at the ‘right time’ (time-of-stress-act), ‘right place’ (tissue-of-act), and ‘right expression’ (time-of-transcription). Heat stress at the reproductive stage impedes pollen viability and stigma receptiveness, affecting the seed set. Prosopis cineraria, the dominant desert-inhabiting legume tree, is heat- and drought-tolerant. The distribution of heat shock elements in a heat-inducible promoter determines the magnitudes of target gene expression in different tissues/organs. Relative expression of P. cineraria heat shock protein 18.2 (PcHsp18.2) in alternate months of 2021 displayed the highest expression in summer. The flowers collected in June, the hottest month (47oC) of 2021, exhibited a high expression of PcHsp18.2. The germination of the pollen collected was 80%, and the trees eventually set seeds. Comprehensive analysis of the promoter (pPcHSP18.2) and terminator (tPchsp18.2) of PcHsp18.2 by expressing the gusA in tobacco exhibited the highest expression under heat stress as similar to the expression of PcHsp18.2 in environmental samples. Ectopic expression of gusA under PcHsp18.2 promoter and terminator resulted in an increased seed set due to the viability of pollen and stigma under heat stress. The gusA expression under PcHsp18.2 promoter and terminator was high-fold in anther compared to the Lat52 and g10 (endogenous genes under its promoter and terminator) genes under heat stress. The expression of genes under strong and balanced endogenous inducible promoter and terminator combinations, as in the desert-growing P. cineraria in transgenic plants, enables the development of resilient crops.

Characterizing a lethal CAG-ACE2 transgenic mouse model for SARS-CoV-2 infection using Cas9-enhanced nanopore sequencing.

The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the common animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1-1.0 TCID50). Infected mice from the two lines began to show COVID-19 clinical signs three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Metabolic engineering of Escherichia coli for de novo synthesis of 1,4-butanediol

1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltAR163L) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA, car, and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.

Efficient biosynthesis of guanidoacetic acid by a recombinant strain of Bacillus subtilis

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.

Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.

Identification of an Endogenous Strong Promoter in Burkholderia sp. JP2-270.

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, Php, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter Php was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40-80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains.

Enhanced Production and Therapeutic Evaluation of TP4 Peptide in Pichia pastoris Against Hepatocellular Carcinoma HUH-7 Cells

Tilapia piscidin 4 (TP4), a cationic antimicrobial peptide, is recognized for its diverse biological roles, including antibacterial, wound-healing, and anticancer properties. Herein, the codon-optimized sequence of TP4 peptide was expressed using the pPICZαA expression vector containing the AOX1 promoter, a strong and inducible promoter, in the Pichia pastoris KM71H expression system. Recombinant TP4 peptide was purified by Ni-NTA affinity chromatography. After purification, the anticancer activity of TP4 was assessed in HUH-7 hepatocellular carcinoma cells, and the underlying mechanisms were determined. In the present study, it was demonstrated for the first time that recombinant TP4 displayed strong anticancer activity in the human HUH-7 cell line. The TP4 antimicrobial peptide can be used as a competitive candidate for the treatment of cancer cells due to its anticancer effects.

Optimization of a DiCre recombinase system with reduced leakage for conditional genome editing of Cryptosporidium

BackgroundThe dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes.MethodsBased on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses.ResultsThe use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35–75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies.ConclusionsA DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.Graphical

Haplotype analysis identifies functional elements in monoclonal gammopathy of unknown significance

Genome-wide association studies (GWASs) based on common single nucleotide polymorphisms (SNPs) have identified several loci associated with the risk of monoclonal gammopathy of unknown significance (MGUS), a precursor condition for multiple myeloma (MM). We hypothesized that analyzing haplotypes might be more useful than analyzing individual SNPs, as it could identify functional chromosomal units that collectively contribute to MGUS risk. To test this hypothesis, we used data from our previous GWAS on 992 MGUS cases and 2910 controls from three European populations. We identified 23 haplotypes that were associated with the risk of MGUS at the genome-wide significance level (p < 5 × 10−8) and showed consistent results among all three populations. In 10 genomic regions, strong promoter, enhancer and regulatory element-related histone marks and their connections to target genes as well as genome segmentation data supported the importance of these regions in MGUS susceptibility. Several associated haplotypes affected pathways important for MM cell survival such as ubiquitin-proteasome system (RNF186, OTUD3), PI3K/AKT/mTOR (HINT3), innate immunity (SEC14L1, ZBP1), cell death regulation (BID) and NOTCH signaling (RBPJ). These pathways are important current therapeutic targets for MM, which may highlight the advantage of the haplotype approach homing to functional units.