Pub Date : 2024-06-28eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12132
Pan Zhang, Biliang Zhang, Yuan-Yuan Ji, Jian Jiao, Ziding Zhang, Chang-Fu Tian
Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.
{"title":"Cofitness network connectivity determines a fuzzy essential zone in open bacterial pangenome.","authors":"Pan Zhang, Biliang Zhang, Yuan-Yuan Ji, Jian Jiao, Ziding Zhang, Chang-Fu Tian","doi":"10.1002/mlf2.12132","DOIUrl":"10.1002/mlf2.12132","url":null,"abstract":"<p><p>Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for <i>Sinorhizobium</i> strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in <i>Sinorhizobium</i> pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"277-290"},"PeriodicalIF":4.5,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211677/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The microbial synthesis of sulfonolipids within the human body is likely involved in maintaining human health or causing diseases. However, the enzymes responsible for their biosynthesis remain largely unknown. In this study, we identified and verified the role of 3-ketocapnine reductase, the third-step enzyme, in the four-step conversion of l-phosphoserine into sulfobacin B both in vivo and in vitro. This finding builds upon our previous research into sulfonolipid biosynthesis, which focused on the vaginal bacterium Chryseobacterium gleum DSM 16776 and the gut bacterium Alistipes finegoldii DSM 17242. Through comprehensive gene mapping, we demonstrate the widespread presence of potential sulfonolipid biosynthetic genes across diverse bacterial species inhabiting various regions of the human body. These findings shed light on the prevalence of sulfonolipid-like metabolites within the human microbiota, suggesting a potential role for these lipid molecules in influencing the intricate biointeractions within the complex microbial ecosystem of the human body.
{"title":"Identification of 3-ketocapnine reductase activity within the human microbiota.","authors":"Xiaotong Wu, Lukuan Hou, Haili Zhang, Yi Ma, Jufang Wang, Mingwei Cai, Xiaoyu Tang","doi":"10.1002/mlf2.12134","DOIUrl":"10.1002/mlf2.12134","url":null,"abstract":"<p><p>The microbial synthesis of sulfonolipids within the human body is likely involved in maintaining human health or causing diseases. However, the enzymes responsible for their biosynthesis remain largely unknown. In this study, we identified and verified the role of 3-ketocapnine reductase, the third-step enzyme, in the four-step conversion of l-phosphoserine into sulfobacin B both in vivo and in vitro. This finding builds upon our previous research into sulfonolipid biosynthesis, which focused on the vaginal bacterium <i>Chryseobacterium gleum</i> DSM 16776 and the gut bacterium <i>Alistipes finegoldii</i> DSM 17242. Through comprehensive gene mapping, we demonstrate the widespread presence of potential sulfonolipid biosynthetic genes across diverse bacterial species inhabiting various regions of the human body. These findings shed light on the prevalence of sulfonolipid-like metabolites within the human microbiota, suggesting a potential role for these lipid molecules in influencing the intricate biointeractions within the complex microbial ecosystem of the human body.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"307-316"},"PeriodicalIF":4.5,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211663/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-28eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12126
Ching Tse, Kesen Ma
Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in H. butylicus to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of H. butylicus, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H. butylicus. HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as kcat/Km, with kcat and apparent Km values of 8.00 ± 0.22 s-1 and 0.59 ± 0.07 mM, respectively. The apparent Km values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in H. butylicus. The putative conserved motif sites for NAD(P)+ and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.
丁醇嗜热菌(Hyperthermus butylicus)是一种嗜热栗色菌,其最终产物为 1-丁醇。丁醇嗜热菌中必须存在一种可恒温的醇脱氢酶(ADH),它是产生这种产物的关键酶;然而,编码 ADH 的基因尚未确定。我们从丁酸梭菌的无细胞提取物中纯化出了一种新型 ADH--HbADH2,并确定了它的特征。编码 HbADH2 的基因被证明是 HBUT_RS04850,并被注释为丁酸杆菌中的一种假定蛋白。研究发现,HbADH2 是一种初级-次级 ADH,能够使用多种底物,包括丁醛和丁醇。丁醛的特异性常数最高,以 k c at/K m 计算,k cat 和表观 K m 值分别为 8.00 ± 0.22 s-1 和 0.59 ± 0.07 mM。其他底物(包括乙醇、1-丙醇、2-丙醇、丁醇、乙醛、丙醛和丙酮)的表观 K m 值分别为 4.36 ± 0.42、4.69 ± 0.41、3.74 ± 0.46、2.44 ± 0.30、1.27 ± 0.18、1.55 ± 0.20 和 0.68 ± 0.04 mM。催化醛还原和醇氧化的最佳 pH 值分别为 6.0 和 9.0,而最佳温度则高于 90°C,这是因为酶活性从 60°C 升高到 90°C。根据其底物特异性、酶动力学和恒温性,HbADH2 可能是催化丁酸杆菌产生 1-丁醇的 ADH。通过将 HbADH2 与先前表征的含铁 ADH 进行比对,确定了 NAD(P)+ 和铁结合的推定保守基团位点。
{"title":"A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon <i>Hyperthermus butylicus</i>.","authors":"Ching Tse, Kesen Ma","doi":"10.1002/mlf2.12126","DOIUrl":"10.1002/mlf2.12126","url":null,"abstract":"<p><p><i>Hyperthermus butylicus</i> is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in <i>H. butylicus</i> to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of <i>H. butylicus</i>, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be <i>HBUT_RS04850</i> and annotated as a hypothetical protein in <i>H. butylicus</i>. HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as <i>k</i> <sub>c</sub> <sub>at</sub>/<i>K</i> <sub>m</sub>, with <i>k</i> <sub>cat</sub> and apparent <i>K</i> <sub>m</sub> values of 8.00 ± 0.22 s<sup>-1</sup> and 0.59 ± 0.07 mM, respectively. The apparent <i>K</i> <sub>m</sub> values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in <i>H. butylicus</i>. The putative conserved motif sites for NAD(P)<sup>+</sup> and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"317-325"},"PeriodicalIF":4.5,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211662/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-28eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12133
Di Wang, Toshiyuki Ueki, Peiyu Ma, Dake Xu, Derek R Lovley
Sulfate-reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe0 corrosion with Desulfovibrio vulgaris, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe0 as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe0 was corroded in cultures of a D. vulgaris hydrogenase-deficient mutant with the 1:1 correspondence between Fe0 loss and H2 accumulation expected for Fe0 oxidation coupled to H+ reduction to H2. This result and the extent of sulfate reduction indicated that D. vulgaris was not capable of direct Fe0-to-microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase-deficient mutant cultures was greater than in sterile controls, demonstrating that H2 removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H2-consuming strain corroded more Fe0 than the mutant strain, which could be attributed to H2 oxidation coupled to sulfate reduction, producing sulfide that further stimulated Fe0 oxidation. The results suggest that H2 consumption is not necessary for microbially enhanced corrosion, but H2 oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that D. vulgaris was incapable of direct electron uptake from Fe0 reaffirms that direct metal-to-microbe electron transfer has yet to be rigorously described in sulfate-reducing microbes.
{"title":"Elucidating microbial iron corrosion mechanisms with a hydrogenase-deficient strain of <i>Desulfovibrio vulgaris</i>.","authors":"Di Wang, Toshiyuki Ueki, Peiyu Ma, Dake Xu, Derek R Lovley","doi":"10.1002/mlf2.12133","DOIUrl":"10.1002/mlf2.12133","url":null,"abstract":"<p><p>Sulfate-reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe<sup>0</sup> corrosion with <i>Desulfovibrio vulgaris</i>, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe<sup>0</sup> as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe<sup>0</sup> was corroded in cultures of a <i>D. vulgaris</i> hydrogenase-deficient mutant with the 1:1 correspondence between Fe<sup>0</sup> loss and H<sub>2</sub> accumulation expected for Fe<sup>0</sup> oxidation coupled to H<sup>+</sup> reduction to H<sub>2</sub>. This result and the extent of sulfate reduction indicated that <i>D. vulgaris</i> was not capable of direct Fe<sup>0</sup>-to-microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase-deficient mutant cultures was greater than in sterile controls, demonstrating that H<sub>2</sub> removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H<sub>2</sub>-consuming strain corroded more Fe<sup>0</sup> than the mutant strain, which could be attributed to H<sub>2</sub> oxidation coupled to sulfate reduction, producing sulfide that further stimulated Fe<sup>0</sup> oxidation. The results suggest that H<sub>2</sub> consumption is not necessary for microbially enhanced corrosion, but H<sub>2</sub> oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that <i>D. vulgaris</i> was incapable of direct electron uptake from Fe<sup>0</sup> reaffirms that direct metal-to-microbe electron transfer has yet to be rigorously described in sulfate-reducing microbes.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"269-276"},"PeriodicalIF":4.5,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211667/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-27eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12123
Tao Wang, Peng Tan, Qi Tang, Chenlong Zhou, Yakun Ding, Shenrui Xu, Mengda Song, Huiyang Fu, Yucheng Zhang, Xiaohui Zhang, Yueyu Bai, Zhihong Sun, Xi Ma
Broad-spectrum antibacterial drugs often lack specificity, leading to indiscriminate bactericidal activity, which can disrupt the normal microbial balance of the host flora and cause unnecessary cytotoxicity during systemic administration. In this study, we constructed a specifically targeted antimicrobial peptide against Staphylococcus aureus by introducing a phage-displayed peptide onto a broad-spectrum antimicrobial peptide and explored its structure-function relationship through one-factor modification. SFK2 obtained by screening based on the selectivity index and the targeting index showed specific killing ability against S. aureus. Moreover, SFK2 showed excellent biocompatibility in mice and piglet, and demonstrated significant therapeutic efficacy against S. aureus infection. In conclusion, our screening of phage-derived heptapeptides effectively enhances the specific bactericidal ability of the antimicrobial peptides against S. aureus, providing a theoretical basis for developing targeted antimicrobial peptides.
{"title":"Phage-displayed heptapeptide sequence conjugation significantly improves the specific targeting ability of antimicrobial peptides against <i>Staphylococcus aureus</i>.","authors":"Tao Wang, Peng Tan, Qi Tang, Chenlong Zhou, Yakun Ding, Shenrui Xu, Mengda Song, Huiyang Fu, Yucheng Zhang, Xiaohui Zhang, Yueyu Bai, Zhihong Sun, Xi Ma","doi":"10.1002/mlf2.12123","DOIUrl":"10.1002/mlf2.12123","url":null,"abstract":"<p><p>Broad-spectrum antibacterial drugs often lack specificity, leading to indiscriminate bactericidal activity, which can disrupt the normal microbial balance of the host flora and cause unnecessary cytotoxicity during systemic administration. In this study, we constructed a specifically targeted antimicrobial peptide against <i>Staphylococcus aureus</i> by introducing a phage-displayed peptide onto a broad-spectrum antimicrobial peptide and explored its structure-function relationship through one-factor modification. SFK2 obtained by screening based on the selectivity index and the targeting index showed specific killing ability against <i>S. aureus</i>. Moreover, SFK2 showed excellent biocompatibility in mice and piglet, and demonstrated significant therapeutic efficacy against <i>S. aureus</i> infection. In conclusion, our screening of phage-derived heptapeptides effectively enhances the specific bactericidal ability of the antimicrobial peptides against <i>S. aureus</i>, providing a theoretical basis for developing targeted antimicrobial peptides.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"251-268"},"PeriodicalIF":4.5,"publicationDate":"2024-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211671/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-27eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12118
Yunxuan Guan, Di Wu, Hui Wang, Ning-Ning Liu
Human microbiomes, considered as a new emerging and enabling cancer hallmark, are increasingly recognized as critical effectors in cancer development and progression. Manipulation of microbiome revitalizing anticancer therapy from natural products shows promise toward improving cancer outcomes. Herein, we summarize our current understanding of the human microbiome-driven molecular mechanisms impacting cancer progression and anticancer therapy. We highlight the potential translational and clinical implications of natural products for cancer prevention and treatment by developing targeted therapeutic strategies as adjuvants for chemotherapy and immunotherapy against tumorigenesis. The challenges and opportunities for future investigations using modulation of the microbiome for cancer treatment are further discussed in this review.
{"title":"Microbiome-driven anticancer therapy: A step forward from natural products.","authors":"Yunxuan Guan, Di Wu, Hui Wang, Ning-Ning Liu","doi":"10.1002/mlf2.12118","DOIUrl":"10.1002/mlf2.12118","url":null,"abstract":"<p><p>Human microbiomes, considered as a new emerging and enabling cancer hallmark, are increasingly recognized as critical effectors in cancer development and progression. Manipulation of microbiome revitalizing anticancer therapy from natural products shows promise toward improving cancer outcomes. Herein, we summarize our current understanding of the human microbiome-driven molecular mechanisms impacting cancer progression and anticancer therapy. We highlight the potential translational and clinical implications of natural products for cancer prevention and treatment by developing targeted therapeutic strategies as adjuvants for chemotherapy and immunotherapy against tumorigenesis. The challenges and opportunities for future investigations using modulation of the microbiome for cancer treatment are further discussed in this review.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"219-230"},"PeriodicalIF":4.5,"publicationDate":"2024-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211674/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-02eCollection Date: 2024-06-01DOI: 10.1002/mlf2.12119
Chenjun Guo, Zixuan Wang, Ji-Long Liu
Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
{"title":"Filamentation and inhibition of prokaryotic CTP synthase with ligands.","authors":"Chenjun Guo, Zixuan Wang, Ji-Long Liu","doi":"10.1002/mlf2.12119","DOIUrl":"10.1002/mlf2.12119","url":null,"abstract":"<p><p>Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of <i>Escherichia coli</i> CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 2","pages":"240-250"},"PeriodicalIF":4.5,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211670/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141474095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-22eCollection Date: 2024-03-01DOI: 10.1002/mlf2.12115
Pingping Wu, Wenjuan Mo, Tian Tian, Kunfeng Song, Yilin Lyu, Haiyan Ren, Jungang Zhou, Yao Yu, Hong Lu
Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.
马氏酵母(Kluyveromyces marxianus)是一种食品安全酵母,在生产异源蛋白方面具有巨大潜力。提高 K. marxianus 的产量仍然是一个挑战,而整合大规模功能模块则是工程中的一个技术障碍。为了解决这些问题,我们构建了马钱子酵母的线性和环状酵母人工染色体(KmYACs),并加载了来自 Pichia pastoris 或马钱子酵母的二硫键形成模块。这些模块最多包含 7 个基因,最大大小为 15 kb。KmYACs 带有来自 K. marxianus 或四膜虫的端粒。无论端粒的类型和 KmYACs 的配置如何,KmYACs 都能成功转移到 K. marxianus 中,并在不影响宿主正常生长的情况下稳定繁殖。KmYACs 提高了二硫键形成基因的整体表达水平,并显著提高了各种异源蛋白的产量。在高密度发酵中,使用 KmYACs 可使葡萄糖淀粉酶产量达到 16.8 克/升,这是迄今为止所报道的 K. marxianus 的最高水平。对含有 KmYACs 的细胞进行的转录组和代谢组分析表明,黄素腺嘌呤二核苷酸生物合成增加、进入三羧酸循环的通量增加,以及对赖氨酸和精氨酸的优先需求是过表达异源蛋白细胞的特征。同样,补充赖氨酸或精氨酸可进一步提高产量。因此,KmYAC 为操作大型模块提供了一个强大的平台,在工业应用和基础研究方面具有巨大潜力。事实证明,通过YACs转移二硫键形成模块是提高异源蛋白产量的有效策略,这一策略可用于优化其他微生物细胞工厂。
{"title":"Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in <i>Kluyveromyces marxianus</i>.","authors":"Pingping Wu, Wenjuan Mo, Tian Tian, Kunfeng Song, Yilin Lyu, Haiyan Ren, Jungang Zhou, Yao Yu, Hong Lu","doi":"10.1002/mlf2.12115","DOIUrl":"10.1002/mlf2.12115","url":null,"abstract":"<p><p><i>Kluyveromyces marxianus</i> is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in <i>K. marxianus</i> remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of <i>K. marxianus</i> (KmYACs) were constructed and loaded with disulfide bond formation modules from <i>Pichia pastoris</i> or <i>K. marxianus</i>. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from <i>K. marxianus</i> or <i>Tetrahymena</i>. KmYACs were transferred successfully into <i>K. marxianus</i> and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in <i>K. marxianus</i>. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":"3 1","pages":"129-142"},"PeriodicalIF":0.0,"publicationDate":"2024-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139206/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141201209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}