Pub Date : 2024-09-28DOI: 10.1186/s12934-024-02524-9
Xiaofang Yan, Anqi Bu, Yanfei Yuan, Xin Zhang, Zhanglin Lin, Xiaofeng Yang
Background: Microbial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner.
Results: In this study, we aimed to enhance the robustness and productivity of Escherichia coli, a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter PesaS and stepwise evaluation, we developed an optimal Esa-PBD(L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8.
Conclusions: This study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.
{"title":"Engineering quorum sensing-based genetic circuits enhances growth and productivity robustness of industrial E. coli at low pH.","authors":"Xiaofang Yan, Anqi Bu, Yanfei Yuan, Xin Zhang, Zhanglin Lin, Xiaofeng Yang","doi":"10.1186/s12934-024-02524-9","DOIUrl":"https://doi.org/10.1186/s12934-024-02524-9","url":null,"abstract":"<p><strong>Background: </strong>Microbial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner.</p><p><strong>Results: </strong>In this study, we aimed to enhance the robustness and productivity of Escherichia coli, a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter P<sub>esaS</sub> and stepwise evaluation, we developed an optimal Esa-P<sub>BD</sub>(L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8.</p><p><strong>Conclusions: </strong>This study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"256"},"PeriodicalIF":4.3,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11438209/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142349886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bionanofertilizers are promising eco-friendly alternative to chemical fertilizers, leveraging nanotechnology and biotechnology to enhance nutrient uptake by plants and improve soil health. They consist of nanoscale materials and beneficial microorganisms, offering benefits such as enhanced seed germination, improved soil quality, increased nutrient use efficiency, and pesticide residue degradation, ultimately leading to improved crop productivity. Bionanofertilizers are designed for targeted delivery of nutrients, controlled release, and minimizing environmental pollutants, making them a sustainable option for agriculture. These fertilizers also have the potential to enhance plant growth, provide disease resistance, and contribute to sustainable farming practices. The development of bionanofertilizers addresses the adverse environmental impact of chemical fertilizers, offering a safer and productive means of fertilization for agricultural practices. This review provides substantial evidence supporting the potential of bionanofertilizers in revolutionizing agricultural practices, offering eco-friendly and sustainable solutions for crop management and soil health.
{"title":"Next-generation fertilizers: the impact of bionanofertilizers on sustainable agriculture.","authors":"Pankaj Kumar Arora, Shivam Tripathi, Rishabh Anand Omar, Prerna Chauhan, Vijay Kumar Sinhal, Amit Singh, Alok Srivastava, Sanjay Kumar Garg, Vijay Pal Singh","doi":"10.1186/s12934-024-02528-5","DOIUrl":"https://doi.org/10.1186/s12934-024-02528-5","url":null,"abstract":"<p><p>Bionanofertilizers are promising eco-friendly alternative to chemical fertilizers, leveraging nanotechnology and biotechnology to enhance nutrient uptake by plants and improve soil health. They consist of nanoscale materials and beneficial microorganisms, offering benefits such as enhanced seed germination, improved soil quality, increased nutrient use efficiency, and pesticide residue degradation, ultimately leading to improved crop productivity. Bionanofertilizers are designed for targeted delivery of nutrients, controlled release, and minimizing environmental pollutants, making them a sustainable option for agriculture. These fertilizers also have the potential to enhance plant growth, provide disease resistance, and contribute to sustainable farming practices. The development of bionanofertilizers addresses the adverse environmental impact of chemical fertilizers, offering a safer and productive means of fertilization for agricultural practices. This review provides substantial evidence supporting the potential of bionanofertilizers in revolutionizing agricultural practices, offering eco-friendly and sustainable solutions for crop management and soil health.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"23 1","pages":"254"},"PeriodicalIF":4.3,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11414052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-19DOI: 10.1186/s12934-024-02522-x
Stefan W. Ritter, Quentin P. Thiel, Martina I. Gastl, Thomas M. Becker
The market for beverages is highly changing within the last years. Increasing consumer awareness towards healthier drinks led to the revival of traditional and the creation of innovative beverages. Various protein-rich legumes were used for milk analogues, which might be also valuable raw materials for refreshing, protein-rich beverages. However, no such applications have been marketed so far, which might be due to unpleasant organoleptic impressions like the legume-typical “beany” aroma. Lactic acid fermentation has already been proven to be a remedy to overcome this hindrance in consumer acceptance. In this study, a statistically based approach was used to elucidate the impact of the fermentation parameters temperature, inoculum cell concentration, and methionine addition on the fermentation of lupine- and faba bean-based substrates. A total of 39 models were found and verified. The majority of these models indicate a strong impact of the temperature on the reduction of aldehydes connected to the “beany” impression (e.g., hexanal) and on the production of pleasantly perceived aroma compounds (e.g., β-damascenone). Positively, the addition of methionine had only minor impacts on the negatively associated sulfuric compounds methional, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Moreover, in further fermentations, the time was added as an additional parameter. It was shown that the strains grew well, strongly acidified the both substrates (pH ≤ 4.0) within 6.5 h, and reached cell counts of > 9 log10 CFU/mL after 24 h. Notably, most of the aldehydes (like hexanal) were reduced within the first 6–7 h, whereas pleasant compounds like β-damascenone reached high concentrations especially in the later fermentation (approx. 24–48 h). Out of the fermentation parameters temperature, inoculum cell concentration, and methionine addition, the temperature had the highest influence on the observed aroma and taste active compounds. As the addition of methionine to compensate for the legume-typical deficit did not lead to an adverse effect, fortifying legume-based substrates with methionine should be considered to improve the bioavailability of the legume protein. Aldehydes, which are associated with the “beany” aroma impression, can be removed efficiently in fermentation. However, terminating the process prematurely would lead to an incomplete production of pleasant aroma compounds.
{"title":"Optimizing the fermentation parameters in the Lactic Acid Fermentation of Legume-based Beverages– a statistically based fermentation","authors":"Stefan W. Ritter, Quentin P. Thiel, Martina I. Gastl, Thomas M. Becker","doi":"10.1186/s12934-024-02522-x","DOIUrl":"https://doi.org/10.1186/s12934-024-02522-x","url":null,"abstract":"The market for beverages is highly changing within the last years. Increasing consumer awareness towards healthier drinks led to the revival of traditional and the creation of innovative beverages. Various protein-rich legumes were used for milk analogues, which might be also valuable raw materials for refreshing, protein-rich beverages. However, no such applications have been marketed so far, which might be due to unpleasant organoleptic impressions like the legume-typical “beany” aroma. Lactic acid fermentation has already been proven to be a remedy to overcome this hindrance in consumer acceptance. In this study, a statistically based approach was used to elucidate the impact of the fermentation parameters temperature, inoculum cell concentration, and methionine addition on the fermentation of lupine- and faba bean-based substrates. A total of 39 models were found and verified. The majority of these models indicate a strong impact of the temperature on the reduction of aldehydes connected to the “beany” impression (e.g., hexanal) and on the production of pleasantly perceived aroma compounds (e.g., β-damascenone). Positively, the addition of methionine had only minor impacts on the negatively associated sulfuric compounds methional, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Moreover, in further fermentations, the time was added as an additional parameter. It was shown that the strains grew well, strongly acidified the both substrates (pH ≤ 4.0) within 6.5 h, and reached cell counts of > 9 log10 CFU/mL after 24 h. Notably, most of the aldehydes (like hexanal) were reduced within the first 6–7 h, whereas pleasant compounds like β-damascenone reached high concentrations especially in the later fermentation (approx. 24–48 h). Out of the fermentation parameters temperature, inoculum cell concentration, and methionine addition, the temperature had the highest influence on the observed aroma and taste active compounds. As the addition of methionine to compensate for the legume-typical deficit did not lead to an adverse effect, fortifying legume-based substrates with methionine should be considered to improve the bioavailability of the legume protein. Aldehydes, which are associated with the “beany” aroma impression, can be removed efficiently in fermentation. However, terminating the process prematurely would lead to an incomplete production of pleasant aroma compounds.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"34 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142269517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1186/s12934-024-02516-9
Eun Jung Jeon, Seong Min Lee, Hee Soo Hong, Ki Jun Jeong
Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging. We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for ⍺-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results. By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.
{"title":"Design of fully synthetic signal peptide library and its use for enhanced secretory production of recombinant proteins in Corynebacterium glutamicum","authors":"Eun Jung Jeon, Seong Min Lee, Hee Soo Hong, Ki Jun Jeong","doi":"10.1186/s12934-024-02516-9","DOIUrl":"https://doi.org/10.1186/s12934-024-02516-9","url":null,"abstract":"Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging. We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for ⍺-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results. By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"199 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142269625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1186/s12934-024-02501-2
Yinlu Liu, Qianzhen Dong, Wan Song, Wenwen Pei, Yan Zeng, Min Wang, Yuanxia Sun, Yanhe Ma, Jiangang Yang
Seven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production. In this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production. We achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.
{"title":"Microbial synthesis of sedoheptulose from glucose by metabolically engineered Corynebacterium glutamicum","authors":"Yinlu Liu, Qianzhen Dong, Wan Song, Wenwen Pei, Yan Zeng, Min Wang, Yuanxia Sun, Yanhe Ma, Jiangang Yang","doi":"10.1186/s12934-024-02501-2","DOIUrl":"https://doi.org/10.1186/s12934-024-02501-2","url":null,"abstract":"Seven-carbon sugars, which rarely exist in nature, are the key constitutional unit of septacidin and hygromycin B in bacteria. These sugars exhibit a potential therapeutic effect for hypoglycaemia and cancer and serve as building blocks for the synthesis of C-glycosides and novel antibiotics. However, chemical and enzymatic approaches for the synthesis of seven-carbon sugars have faced challenges, such as complex reaction steps, low overall yields and high-cost feedstock, limiting their industrial-scale production. In this work, we propose a strain engineering approach for synthesising sedoheptulose using glucose as sole feedstock. The gene pfkA encoding 6-phosphofructokinase in Corynebacterium glutamicum was inactivated to direct the carbon flux towards the pentose phosphate pathway in the cellular metabolic network. This genetic modification successfully enabled the synthesis of sedoheptulose from glucose. Additionally, we identified key enzymes responsible for product formation through transcriptome analysis, and their corresponding genes were overexpressed, resulting in a further 20% increase in sedoheptulose production. We achieved a sedoheptulose concentration of 24 g/L with a yield of 0.4 g/g glucose in a 1 L fermenter, marking the highest value up to date. The produced sedoheptulose could further function as feedstock for synthesising structural seven-carbon sugars through coupling with enzymatic isomerisation, epimerisation and reduction reactions.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"16 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1186/s12934-024-02518-7
Yasmine Fathy Mohamed, Rachel C. Fernandez
Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.
百日咳杆菌是百日咳或百日咳的病原体。虽然无细胞百日咳疫苗（aP）和全细胞百日咳疫苗（wP）都能预防疾病，但高致反应性的全细胞百日咳疫苗能更好地预防定植和传播。致反应性主要归因于百日咳杆菌脂寡糖（LOS）中的脂质 A 分子。在 LOS 中，脂质 A 充当疏水锚，与宿主免疫细胞上的 TLR4-MD2 结合，启动 MyD88 依赖性和 TRIF 依赖性途径，从而影响适应性免疫反应。脂质 A 的变体，如单磷脂质 A（MPLA），也可以作为佐剂。佐剂可以克服 aP 疫苗的缺点。这项研究利用来自其他细菌的脂质 A 修饰酶，在百日咳杆菌中产生了一种类似 MPLA 的佐剂菌株。我们创建了具有不同脂质 A 修饰的百日咳杆菌菌株，并使用 MALDI-TOF 对其进行了验证。我们设计了一种六烷基化的单磷酸化脂质 A，它能显著降低人类 TLR4 的活化并激活 TRIF 通路。经修饰的脂寡糖（LOS）可促进 IRF3 磷酸化和 I 型干扰素的产生，与 MPLA 反应类似。我们还生成了另外三种变体，它们具有更强的佐剂特性和更低的内毒素。使用单核细胞活化试验（MAT）进行的热原性研究表明，这四种脂质 A 变体能显著降低外周血单核细胞（PBMCs）中作为发热标志的 IL-6 的反应。这些发现为开发致反应性可能较低的 wP 疫苗以及为现有疫苗配方设计适应性强的佐剂铺平了道路，从而推进了更有效的百日咳免疫策略。
{"title":"Programming Bordetella pertussis lipid A to promote adjuvanticity","authors":"Yasmine Fathy Mohamed, Rachel C. Fernandez","doi":"10.1186/s12934-024-02518-7","DOIUrl":"https://doi.org/10.1186/s12934-024-02518-7","url":null,"abstract":"Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"32 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1186/s12934-024-02523-w
Jasmine De Baets, Brecht De Paepe, Marjan De Mey
Engineering bacteria with the purpose of optimizing the production of interesting molecules often leads to a decrease in growth due to metabolic burden or toxicity. By delaying the production in time, these negative effects on the growth can be avoided in a process called a two-stage fermentation. During this two-stage fermentation process, the production stage is only activated once sufficient cell mass is obtained. Besides the possibility of using external triggers, such as chemical molecules or changing fermentation parameters to induce the production stage, there is a renewed interest towards autoinducible systems. These systems, such as quorum sensing, do not require the extra interference with the fermentation broth to start the induction. In this review, we discuss the different possibilities of both external and autoinduction methods to obtain a two-stage fermentation. Additionally, an overview is given of the tuning methods that can be applied to optimize the induction process. Finally, future challenges and prospects of (auto)inducible expression systems are discussed. There are numerous methods to obtain a two-stage fermentation process each with their own advantages and disadvantages. Even though chemically inducible expression systems are well-established, an increasing interest is going towards autoinducible expression systems, such as quorum sensing. Although these newer techniques cannot rely on the decades of characterization and applications as is the case for chemically inducible promoters, their advantages might lead to a shift in future inducible expression systems.
{"title":"Delaying production with prokaryotic inducible expression systems","authors":"Jasmine De Baets, Brecht De Paepe, Marjan De Mey","doi":"10.1186/s12934-024-02523-w","DOIUrl":"https://doi.org/10.1186/s12934-024-02523-w","url":null,"abstract":"Engineering bacteria with the purpose of optimizing the production of interesting molecules often leads to a decrease in growth due to metabolic burden or toxicity. By delaying the production in time, these negative effects on the growth can be avoided in a process called a two-stage fermentation. During this two-stage fermentation process, the production stage is only activated once sufficient cell mass is obtained. Besides the possibility of using external triggers, such as chemical molecules or changing fermentation parameters to induce the production stage, there is a renewed interest towards autoinducible systems. These systems, such as quorum sensing, do not require the extra interference with the fermentation broth to start the induction. In this review, we discuss the different possibilities of both external and autoinduction methods to obtain a two-stage fermentation. Additionally, an overview is given of the tuning methods that can be applied to optimize the induction process. Finally, future challenges and prospects of (auto)inducible expression systems are discussed. There are numerous methods to obtain a two-stage fermentation process each with their own advantages and disadvantages. Even though chemically inducible expression systems are well-established, an increasing interest is going towards autoinducible expression systems, such as quorum sensing. Although these newer techniques cannot rely on the decades of characterization and applications as is the case for chemically inducible promoters, their advantages might lead to a shift in future inducible expression systems.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"19 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1186/s12934-024-02519-6
Juan José León, Nía Oetiker, Nicolás Torres, Nicolás Bruna, Evgenii Oskolkov, Pedro Lei, Andrey N. Kuzmin, Kaiwen Chen, Stelios Andreadis, Blaine A. Pfeifer, Mark T. Swihart, Paras N. Prasad, José Pérez-Donoso
Rare-earth sulfide nanoparticles (NPs) could harness the optical and magnetic features of rare-earth ions for applications in nanotechnology. However, reports of their synthesis are scarce and typically require high temperatures and long synthesis times. Here we present a biosynthesis of terbium sulfide (TbS) NPs using microorganisms, identifying conditions that allow Escherichia coli to extracellularly produce TbS NPs in aqueous media at 37 °C by controlling cellular sulfur metabolism to produce a high concentration of sulfide ions. Electron microscopy revealed ultrasmall spherical NPs with a mean diameter of 4.1 ± 1.3 nm. Electron diffraction indicated a high degree of crystallinity, while elemental mapping confirmed colocalization of terbium and sulfur. The NPs exhibit characteristic absorbance and luminescence of terbium, with downshifting quantum yield (QY) reaching 28.3% and an emission lifetime of ~ 2 ms. This high QY and long emission lifetime is unusual in a neat rare-earth compound; it is typically associated with rare-earth ions doped into another crystalline lattice to avoid non-radiative cross relaxation. This suggests a reduced role of nonradiative processes in these terbium-based NPs. This is, to our knowledge, the first report revealing the advantage of biosynthesis over chemical synthesis for Rare Earth Element (REE) based NPs, opening routes to new REE-based nanocrystals.
{"title":"Microbial green synthesis of luminescent terbium sulfide nanoparticles using E. Coli: a rare earth element detoxification mechanism","authors":"Juan José León, Nía Oetiker, Nicolás Torres, Nicolás Bruna, Evgenii Oskolkov, Pedro Lei, Andrey N. Kuzmin, Kaiwen Chen, Stelios Andreadis, Blaine A. Pfeifer, Mark T. Swihart, Paras N. Prasad, José Pérez-Donoso","doi":"10.1186/s12934-024-02519-6","DOIUrl":"https://doi.org/10.1186/s12934-024-02519-6","url":null,"abstract":"Rare-earth sulfide nanoparticles (NPs) could harness the optical and magnetic features of rare-earth ions for applications in nanotechnology. However, reports of their synthesis are scarce and typically require high temperatures and long synthesis times. Here we present a biosynthesis of terbium sulfide (TbS) NPs using microorganisms, identifying conditions that allow Escherichia coli to extracellularly produce TbS NPs in aqueous media at 37 °C by controlling cellular sulfur metabolism to produce a high concentration of sulfide ions. Electron microscopy revealed ultrasmall spherical NPs with a mean diameter of 4.1 ± 1.3 nm. Electron diffraction indicated a high degree of crystallinity, while elemental mapping confirmed colocalization of terbium and sulfur. The NPs exhibit characteristic absorbance and luminescence of terbium, with downshifting quantum yield (QY) reaching 28.3% and an emission lifetime of ~ 2 ms. This high QY and long emission lifetime is unusual in a neat rare-earth compound; it is typically associated with rare-earth ions doped into another crystalline lattice to avoid non-radiative cross relaxation. This suggests a reduced role of nonradiative processes in these terbium-based NPs. This is, to our knowledge, the first report revealing the advantage of biosynthesis over chemical synthesis for Rare Earth Element (REE) based NPs, opening routes to new REE-based nanocrystals.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"18 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1186/s12934-024-02509-8
Anna Weimer, Laura Pause, Fabian Ries, Michael Kohlstedt, Lorenz Adrian, Jens Krömer, Bin Lai, Christoph Wittmann
Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.
{"title":"Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production","authors":"Anna Weimer, Laura Pause, Fabian Ries, Michael Kohlstedt, Lorenz Adrian, Jens Krömer, Bin Lai, Christoph Wittmann","doi":"10.1186/s12934-024-02509-8","DOIUrl":"https://doi.org/10.1186/s12934-024-02509-8","url":null,"abstract":"Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"157 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1186/s12934-024-02489-9
Sumeeta Kumari, Alka Kumari, Asmita Dhiman, Kanti Nandan Mihooliya, Manoj Raje, G. S. Prasad, Anil Kumar Pinnaka
Sophorolipids are glycolipid biosurfactants with potential antibacterial, antifungal, and anticancer applications, rendering them promising for research. Therefore, this study hypothesizes that sophorolipids may have a notable impact on disrupting membrane integrity and triggering the production of reactive oxygen species, ultimately resulting in the eradication of pathogenic microbes. The current study resulted in the isolation of two Metschnikowia novel yeast strains. Sophorolipids production from these strains reached maximum yields of 23.24 g/l and 21.75 g/l, respectively, at the bioreactors level. Biosurfactants sophorolipids were characterized using FTIR and LC–MS techniques and found to be a mixture of acidic and lactonic forms with molecular weights of m/z 678 and 700. Our research elucidated sophorolipids’ mechanism in disrupting bacterial and fungal membranes through ROS generation, confirmed by transmission electron microscopy and FACS analysis. The results showed that these compounds disrupted the membrane integrity and induced ROS production, leading to cell death in Klebsiella pneumoniae and Fusarium solani. In addition, the anticancer properties of sophorolipids were investigated on the A549 lung cancer cell line and found that sophorolipid-11D (SL-11D) and sophorolipid-11X (SL-11X) disrupted the actin cytoskeleton, as evidenced by immunofluorescence microscopy. The A549 cells were stained with Acridine orange/Ethidium bromide, which showed that they underwent necrosis. This was confirmed by flow cytometric analysis using Annexin/PI staining. The SL-11D and SL-11X molecules exhibited low levels of haemolytic activity and in-vitro cytotoxicity in HEK293, Caco-2, and L929 cell lines. In this work, novel yeast species CIG-11DT and CIG-11XT, isolated from the bee’s gut, produce significant yields of sophorolipids without needing secondary oil sources, indicating a more economical production method. Our research shows that sophorolipids disrupt bacterial and fungal membranes via ROS production. They suggest they may act as chemo-preventive agents by inducing apoptosis in lung cancer cells, offering the potential for enhancing anticancer therapies.
{"title":"Unveiling the potential of novel Metschnikowia yeast biosurfactants: triggering oxidative stress for promising antifungal and anticancer activity","authors":"Sumeeta Kumari, Alka Kumari, Asmita Dhiman, Kanti Nandan Mihooliya, Manoj Raje, G. S. Prasad, Anil Kumar Pinnaka","doi":"10.1186/s12934-024-02489-9","DOIUrl":"https://doi.org/10.1186/s12934-024-02489-9","url":null,"abstract":"Sophorolipids are glycolipid biosurfactants with potential antibacterial, antifungal, and anticancer applications, rendering them promising for research. Therefore, this study hypothesizes that sophorolipids may have a notable impact on disrupting membrane integrity and triggering the production of reactive oxygen species, ultimately resulting in the eradication of pathogenic microbes. The current study resulted in the isolation of two Metschnikowia novel yeast strains. Sophorolipids production from these strains reached maximum yields of 23.24 g/l and 21.75 g/l, respectively, at the bioreactors level. Biosurfactants sophorolipids were characterized using FTIR and LC–MS techniques and found to be a mixture of acidic and lactonic forms with molecular weights of m/z 678 and 700. Our research elucidated sophorolipids’ mechanism in disrupting bacterial and fungal membranes through ROS generation, confirmed by transmission electron microscopy and FACS analysis. The results showed that these compounds disrupted the membrane integrity and induced ROS production, leading to cell death in Klebsiella pneumoniae and Fusarium solani. In addition, the anticancer properties of sophorolipids were investigated on the A549 lung cancer cell line and found that sophorolipid-11D (SL-11D) and sophorolipid-11X (SL-11X) disrupted the actin cytoskeleton, as evidenced by immunofluorescence microscopy. The A549 cells were stained with Acridine orange/Ethidium bromide, which showed that they underwent necrosis. This was confirmed by flow cytometric analysis using Annexin/PI staining. The SL-11D and SL-11X molecules exhibited low levels of haemolytic activity and in-vitro cytotoxicity in HEK293, Caco-2, and L929 cell lines. In this work, novel yeast species CIG-11DT and CIG-11XT, isolated from the bee’s gut, produce significant yields of sophorolipids without needing secondary oil sources, indicating a more economical production method. Our research shows that sophorolipids disrupt bacterial and fungal membranes via ROS production. They suggest they may act as chemo-preventive agents by inducing apoptosis in lung cancer cells, offering the potential for enhancing anticancer therapies.","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"3 1","pages":""},"PeriodicalIF":6.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142200159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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