Background: Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC).
Results: In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out.
Conclusions: Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.
{"title":"Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking.","authors":"Víctor Garrigós, Cecilia Picazo, Emilia Matallana, Agustín Aranda","doi":"10.1186/s12934-024-02504-z","DOIUrl":"10.1186/s12934-024-02504-z","url":null,"abstract":"<p><strong>Background: </strong>Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC).</p><p><strong>Results: </strong>In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out.</p><p><strong>Conclusions: </strong>Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11337681/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142009038","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 : 2024-08-16DOI: 10.1186/s12934-024-02495-x
Sara Refaat, Eman Fikry, Nora Tawfeek, Ashraf S A El-Sayed, Maher M El-Domiaty, Azza M El-Shafae
Epothilones are one of the common prescribed anticancer drugs for solid tumors, for their exceptional binding affinity with β-tubulin microtubule, stabilizing their disassembly, causing an ultimate arrest to the cellular growth. Epothilones were initially isolated from Sornagium cellulosum, however, their extremely slow growth rate and low yield of epothilone is the challenge. So, screening for a novel fungal endophyte dwelling medicinal plants, with higher epothilone productivity and feasibility of growth manipulation was the objective. Aspergillus niger EFBL-SR OR342867, an endophyte of Latania loddegesii, has been recognized as the heady epothilone producer (140.2 μg/L). The chemical structural identity of the TLC-purified putative sample of A. niger was resolved from the HPLC, FTIR and LC-ESI-MS/MS analyses, with an identical molecular structure of the authentic epothilone B. The purified A. niger epothilone B showed a resilient activity against MCF-7 (0.022 μM), HepG-2 (0.037 μM), and HCT-116 (0.12 μM), with selectivity indices 21.8, 12.9 and 4, respectively. The purified epothilone B exhibited a potential anti-wound healing activity to HepG-2 and MCF-7 cells by ~ 54.07 and 60.0%, respectively, after 24 h, compared to the untreated cells. The purified epothilone has a significant antiproliferative effect by arresting the cellular growth of MCF-7 at G2/M phase by ~ 2.1 folds, inducing the total apoptosis by ~ 12.2 folds, normalized to the control cells. The epothilone B productivity by A. niger was optimized by the response surface methodology, with ~ 1.4 fold increments (266.9 μg/L), over the control. The epothilone productivity by A. niger was reduced by ~ 2.4 folds by 6 months storage as a slope culture at 4 °C, however, the epothilone productivity was slightly restored with ethylacetate extracts of L. loddegesii, confirming the plant-derived chemical signals that partially triggers the biosynthetic genes of A. niger epothilones. So, this is the first report emphasizing the metabolic potency of A. niger, an endophyte of L. loddegesii, to produce epothilone B, that could be a new platform for industrial production of this drug.
埃博霉素是治疗实体瘤的常用抗癌药物之一,因为它与β-微管蛋白微管有特殊的结合亲和力,能稳定微管的解体,最终导致细胞生长停滞。表霉素最初是从 Sornagium cellulosum 中分离出来的,但其生长速度极慢,表霉素的产量也很低。因此,筛选一种居住在药用植物中的新型真菌内生菌,使其具有更高的表霉素生产率和生长控制的可行性是我们的目标。Latania loddegesii 的内生菌黑曲霉 EFBL-SR OR342867 被认为是一种高效的表皮生长素生产菌(140.2 μg/L)。通过高效液相色谱、傅立叶变换红外光谱和液相色谱-电离串联质谱/质谱分析,确定了经 TLC 纯化的黑僵菌推定样品的化学结构特征,其分子结构与真品表硫酮 B 相同。纯化的 A. niger 表皮霉素 B 对 MCF-7(0.022 μM)、HepG-2(0.037 μM)和 HCT-116(0.12 μM)具有抗性,选择性指数分别为 21.8、12.9 和 4。纯化的埃博霉素 B 对 HepG-2 和 MCF-7 细胞具有潜在的抗伤口愈合活性,与未处理的细胞相比,24 小时后的抗伤口愈合活性分别为 ~ 54.07% 和 60.0%。纯化的埃博霉素具有显著的抗增殖作用,可使 MCF-7 细胞生长在 G2/M 期停止约 2.1 倍,诱导细胞完全凋亡约 12.2 倍(与对照细胞比较)。通过响应面方法优化了黑僵菌的表硫酮 B 生产率,比对照组提高了约 1.4 倍(266.9 μg/L)。在 4 ° C 下斜坡培养 6 个月后,黑僵菌的表硫酮生产率降低了约 2.4 倍,但使用 Loddegesii 的乙酸乙酯提取物后,表硫酮生产率略有恢复,这证实了植物源化学信号部分触发了黑僵菌表硫酮的生物合成基因。因此,这是第一份强调黑僵菌(L. loddegesii 的内生菌)具有生产表皮霉素 B 的代谢能力的报告,这可能是工业化生产这种药物的一个新平台。
{"title":"Production and bioprocessing of epothilone B from Aspergillus niger, an endophyte of Latania loddegesii, with a conceivable biosynthetic stability: anticancer, anti-wound healing activities and cell cycle analysis.","authors":"Sara Refaat, Eman Fikry, Nora Tawfeek, Ashraf S A El-Sayed, Maher M El-Domiaty, Azza M El-Shafae","doi":"10.1186/s12934-024-02495-x","DOIUrl":"10.1186/s12934-024-02495-x","url":null,"abstract":"<p><p>Epothilones are one of the common prescribed anticancer drugs for solid tumors, for their exceptional binding affinity with β-tubulin microtubule, stabilizing their disassembly, causing an ultimate arrest to the cellular growth. Epothilones were initially isolated from Sornagium cellulosum, however, their extremely slow growth rate and low yield of epothilone is the challenge. So, screening for a novel fungal endophyte dwelling medicinal plants, with higher epothilone productivity and feasibility of growth manipulation was the objective. Aspergillus niger EFBL-SR OR342867, an endophyte of Latania loddegesii, has been recognized as the heady epothilone producer (140.2 μg/L). The chemical structural identity of the TLC-purified putative sample of A. niger was resolved from the HPLC, FTIR and LC-ESI-MS/MS analyses, with an identical molecular structure of the authentic epothilone B. The purified A. niger epothilone B showed a resilient activity against MCF-7 (0.022 μM), HepG-2 (0.037 μM), and HCT-116 (0.12 μM), with selectivity indices 21.8, 12.9 and 4, respectively. The purified epothilone B exhibited a potential anti-wound healing activity to HepG-2 and MCF-7 cells by ~ 54.07 and 60.0%, respectively, after 24 h, compared to the untreated cells. The purified epothilone has a significant antiproliferative effect by arresting the cellular growth of MCF-7 at G2/M phase by ~ 2.1 folds, inducing the total apoptosis by ~ 12.2 folds, normalized to the control cells. The epothilone B productivity by A. niger was optimized by the response surface methodology, with ~ 1.4 fold increments (266.9 μg/L), over the control. The epothilone productivity by A. niger was reduced by ~ 2.4 folds by 6 months storage as a slope culture at 4 °C, however, the epothilone productivity was slightly restored with ethylacetate extracts of L. loddegesii, confirming the plant-derived chemical signals that partially triggers the biosynthetic genes of A. niger epothilones. So, this is the first report emphasizing the metabolic potency of A. niger, an endophyte of L. loddegesii, to produce epothilone B, that could be a new platform for industrial production of this drug.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11328370/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996141","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 : 2024-08-16DOI: 10.1186/s12934-024-02506-x
Liyun Ji, Shuo Xu, Yue Zhang, Hairong Cheng
Background: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers.
Results: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter.
Conclusion: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.
背景:非常规酵母菌和细菌在将低成本可再生原料转化为有价值产品方面具有独特的代谢能力,因而在合成生物学中具有重要意义。改进代谢途径和提高生物产品产量仍然依赖于在这些微生物中战略性地使用各种启动子。针对不同宿主开发不同强度的广谱启动子库对生物合成工程师来说很有吸引力:在这项研究中,五个脂肪溶解亚罗酵母组成型启动子(yl.hp4d、yl.FBA1in、yl.TEF1、yl.TDH1、yl.EXP1)和五个马氏克鲁维酵母组成型启动子(km.PDC1、km.FBA1、km.TEF1、km.TDH3、km.ENO1)来构建启动子-报告基因载体,利用α-淀粉酶和红色荧光蛋白(RFP)作为报告基因。我们对脂肪溶解酵母、马克氏酵母、酿酒葡萄孢、大肠杆菌和谷氨酸棒状杆菌的启动子强度进行了系统鉴定。我们发现,五个 K. marxianus 启动子都能在 Y. lipolytica 中表达基因,而五个 Y. lipolytica 启动子都能在 K. marxianus 中表达基因,但表达强度各不相同。值得注意的是,yl.TEF1 和 km.TEF1酵母启动子在P. pastoris、大肠杆菌和谷氨酸酵母中表现出其适应性。在牧马酵母中,yl.TEF1 启动子表现出淀粉酶和 RFP 的大量表达。在大肠杆菌和谷氨酸菌中,真核 km.TEF1 启动子表现出 RFP 的强力表达。值得注意的是,在大肠杆菌中,km.TEF1 启动子的 RFP 表达强度达到了 T7 启动子的 20%:具有多样性和跨领域适用性的非常规酵母启动子在开发创新型动态调控系统方面具有巨大潜力,可有效管理碳通量并提高不同微生物宿主的目标生物产品合成。
{"title":"Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria.","authors":"Liyun Ji, Shuo Xu, Yue Zhang, Hairong Cheng","doi":"10.1186/s12934-024-02506-x","DOIUrl":"10.1186/s12934-024-02506-x","url":null,"abstract":"<p><strong>Background: </strong>Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers.</p><p><strong>Results: </strong>In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter.</p><p><strong>Conclusion: </strong>Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11330142/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996142","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 : 2024-08-14DOI: 10.1186/s12934-024-02500-3
Xuefeng Mei, Deping Hua, Na Liu, Lilin Zhang, Xiaowen Zhao, Yujing Tian, Baiping Zhao, Jinhai Huang, Lei Zhang
Background: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency.
Results: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique.
Conclusions: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.
背景:花青素是植物中的水溶性类黄酮,它赋予植物鲜艳的色彩,被广泛用作食品着色剂、营养素和化妆品添加剂。从植物组织中采集花青素的传统技术存在一些局限性,包括物种、产地、季节和技术等。利用工程微生物生产天然产品的好处包括使用方便、可控性强、效率高:本研究从富含花青素的植物材料蓝莓果实和紫圆茄外皮中成功克隆了十个编码花青素生物合成途径中酶的基因。利用酵母Fab组装技术,在不同启动子下构建了这些基因的转录单元。利用同源重组技术,将 PAL 和 C4H、4CL 和 CHS 的转录单元分别融合并插入到酵母菌株 JDY52 的 XVI 和 IV 链中,得到菌株 A;将含有 CHI 和 F3H、F3'H 和 DFR 转录单元的片段插入到 III 和 XVI 链中,得到菌株 B1。菌株 B2 的染色体 IV 中含有 ANS 和 3GT 的转录单元。通过 LC-MS/MS,利用多菌株共培养技术,按照花青素在 S. cerevisiae 中从头开始生物合成的预测结果,检测到了几种花青素,包括青花素、芍药苷、芍药苷、佩兰苷和麦冬苷:我们提出了一种推进异源从头开始花青素生物合成途径的新概念,并为随后优化花青素的微生物合成提供了基础信息和理论框架。
{"title":"De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry.","authors":"Xuefeng Mei, Deping Hua, Na Liu, Lilin Zhang, Xiaowen Zhao, Yujing Tian, Baiping Zhao, Jinhai Huang, Lei Zhang","doi":"10.1186/s12934-024-02500-3","DOIUrl":"10.1186/s12934-024-02500-3","url":null,"abstract":"<p><strong>Background: </strong>Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency.</p><p><strong>Results: </strong>In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique.</p><p><strong>Conclusions: </strong>We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11323355/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141982701","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 : 2024-08-12DOI: 10.1186/s12934-024-02474-2
Lucija Sovic, Lenny Malihan-Yap, Gábor Szilveszter Tóth, Vilja Siitonen, Véronique Alphand, Yagut Allahverdiyeva, Robert Kourist
The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon. Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm. Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.
氧化还原酶的大规模生物催化应用需要经济高效的氧化还原辅助因子再生系统。这些是工业生物生产的主要瓶颈,也是重要的成本因素。在这项工作中,将 Burkholderia xenovorans 的转化酶基因和 Baeyer-Villiger 单加氧酶基因共同表达到大肠杆菌 W ΔcscR 和大肠杆菌 BL21 (DE3) 中,利用蔗糖作为氧化还原辅助因子再生的电子源,实现了环己酮到聚合物前体ε-己内酯的高效生物转化,转化率与葡萄糖相当。大肠杆菌 W ΔcscR 有一个能利用蔗糖的原生 csc 调节子,它通过删除抑制基因(cscR)而失调,因此即使在浓度低于 6 mM(2 g L-1)时也能吸收蔗糖。另一方面,被广泛用作表达宿主的大肠杆菌 BL21(DE3)不含 csc 调节子。在这里,我们展示了一个概念证明,即两种大肠杆菌宿主共同表达转化酶足以高效利用蔗糖,以维持环己酮拜耳-维里格氧化过程中辅助因子的再生。使用大肠杆菌 W ΔcscR,可获得 37 U gDCW-1 的特异性活性,这表明该菌株适用于重组基因共表达和随后的全细胞生物转化。此外,用大肠杆菌 BL21(DE3)转入相同的共表达盒并进行了研究,结果显示其特异性活性为 17 U gDCW-1。最后,用大肠杆菌 W ΔcscR 表达 BVMO 与来自 Synechocystis S02 的光合蔗糖进行生物转化,结果表明 3 小时后环己酮完全转化,尤其是外质中表达转化酶基因的菌株。研究结果表明,蔗糖可以作为一种替代电子源,驱动重组大肠杆菌菌株的全细胞生物转化,为可持续化学品生产开辟了新策略。
{"title":"Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase","authors":"Lucija Sovic, Lenny Malihan-Yap, Gábor Szilveszter Tóth, Vilja Siitonen, Véronique Alphand, Yagut Allahverdiyeva, Robert Kourist","doi":"10.1186/s12934-024-02474-2","DOIUrl":"https://doi.org/10.1186/s12934-024-02474-2","url":null,"abstract":"The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon. Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm. Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production. ","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141944294","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}
Background: Endophytic fungi (EF) reside within plants without causing harm and provide benefits such as enhancing nutrients and producing bioactive compounds, which improve the medicinal properties of host plants. Selecting plants with established medicinal properties for studying EF is important, as it allows a deeper understanding of their influence. Therefore, the study aimed to investigate the impact of EF after inoculating the medicinal plant Perilla frutescens, specifically focusing on their role in enhancing medicinal properties.
Results: In the current study, the impact of two EF i.e., Irpex lenis and Schizophyllum commune isolated from A. bracteosa was observed on plant Perilla frutescens leaves after inoculation. Plants were divided into four groups i.e., group A: the control group, group B: inoculated with I. lenis; group C: inoculated with S. commune and group D: inoculated with both the EF. Inoculation impact of I. lenis showed an increase in the concentration of chlorophyll a (5.32 mg/g), chlorophyll b (4.46 mg/g), total chlorophyll content (9.78 mg/g), protein (68.517 ± 0.77 mg/g), carbohydrates (137.886 ± 13.71 mg/g), and crude fiber (3.333 ± 0.37%). Furthermore, the plants inoculated with I. lenis showed the highest concentrations of P (14605 mg/kg), Mg (4964.320 mg/kg), Ca (27389.400 mg/kg), and Mn (86.883 mg/kg). The results of the phytochemical analysis also indicated an increased content of total flavonoids (2.347 mg/g), phenols (3.086 mg/g), tannins (3.902 mg/g), and alkaloids (1.037 mg/g) in the leaf extract of P. frutescens inoculated with I. lenis. Thus, overall the best results of inoculation were observed in Group B i.e. inoculated with I. lenis. GC-MS analysis of methanol leaf extract showed ten bioactive constituents, including 9-Octadecenoic acid (Z)-, methyl ester, and hexadecanoic acid, methyl ester as major constituents found in all the groups of P. frutescens leaves. The phenol (gallic acid) and flavonoids (rutin, kaempferol, and quercetin) were also observed to increase after inoculation by HPTLC analysis. The enhancement in the phytochemical content was co-related with improved anti-oxidant potential which was analyzed by DPPH (% Inhibition: 83.45 µg/ml) and FRAP (2.980 µM Fe (II) equivalent) assay as compared with the control group.
Conclusion: Inoculation with I. lenis significantly enhances the uptake of nutritional constituents, phytochemicals, and antioxidant properties in P. frutescens, suggesting its potential to boost the therapeutic properties of host plants.
背景:内生真菌(EF)寄居在植物体内,不会对植物造成危害,还能提供养分和产生生物活性化合物等益处,从而改善寄主植物的药用特性。选择具有既定药用特性的植物来研究内生真菌非常重要,因为这样可以更深入地了解它们的影响。因此,本研究旨在调查接种药用植物紫苏后 EF 的影响,特别关注其在提高药用价值方面的作用:在本研究中,观察了两种 EF(即从 A. bracteosa 分离出的 Irpex lenis 和 Schizophyllum commune)在接种后对植物紫苏叶的影响。植物被分为四组,即 A 组:对照组;B 组:接种了丝核菌;C 组:接种了 S. commune;D 组:接种了两种 EF。从接种影响来看,叶绿素 a(5.32 毫克/克)、叶绿素 b(4.46 毫克/克)、总叶绿素含量(9.78 毫克/克)、蛋白质(68.517 ± 0.77 毫克/克)、碳水化合物(137.886 ± 13.71 毫克/克)和粗纤维(3.333 ± 0.37%)的浓度均有所增加。此外,接种了雷尼氏菌的植株中 P(14605 毫克/千克)、Mg(4964.320 毫克/千克)、Ca(27389.400 毫克/千克)和 Mn(86.883 毫克/千克)的含量最高。植物化学分析结果还表明,接种了莱尼丝藻的 P. frutescens 的叶提取物中总黄酮(2.347 毫克/克)、酚(3.086 毫克/克)、单宁(3.902 毫克/克)和生物碱(1.037 毫克/克)的含量均有所增加。因此,总体而言,B 组(即接种 I. lenis)的接种效果最好。甲醇叶提取物的气相色谱-质谱(GC-MS)分析表明,在所有组的洋二仙草叶中发现了 10 种生物活性成分,包括 9-十八碳烯酸(Z)-,甲酯和十六碳烯酸,甲酯是主要成分。通过 HPTLC 分析,还观察到酚类(没食子酸)和黄酮类(芦丁、山柰酚和槲皮素)在接种后有所增加。与对照组相比,植物化学成分含量的增加与抗氧化潜力的提高有关:接种蛇麻属植物能明显增强洋二仙草(P. frutescens)对营养成分、植物化学物质和抗氧化特性的吸收,这表明蛇麻属植物具有提高宿主植物治疗特性的潜力。
{"title":"Impact of Irpex lenis and Schizophyllum commune endophytic fungi on Perilla frutescens: enhancing nutritional uptake, phytochemicals, and antioxidant potential.","authors":"Kiran Sharma, Rachna Verma, Dinesh Kumar, Vinod Kumar","doi":"10.1186/s12934-024-02491-1","DOIUrl":"10.1186/s12934-024-02491-1","url":null,"abstract":"<p><strong>Background: </strong>Endophytic fungi (EF) reside within plants without causing harm and provide benefits such as enhancing nutrients and producing bioactive compounds, which improve the medicinal properties of host plants. Selecting plants with established medicinal properties for studying EF is important, as it allows a deeper understanding of their influence. Therefore, the study aimed to investigate the impact of EF after inoculating the medicinal plant Perilla frutescens, specifically focusing on their role in enhancing medicinal properties.</p><p><strong>Results: </strong>In the current study, the impact of two EF i.e., Irpex lenis and Schizophyllum commune isolated from A. bracteosa was observed on plant Perilla frutescens leaves after inoculation. Plants were divided into four groups i.e., group A: the control group, group B: inoculated with I. lenis; group C: inoculated with S. commune and group D: inoculated with both the EF. Inoculation impact of I. lenis showed an increase in the concentration of chlorophyll a (5.32 mg/g), chlorophyll b (4.46 mg/g), total chlorophyll content (9.78 mg/g), protein (68.517 ± 0.77 mg/g), carbohydrates (137.886 ± 13.71 mg/g), and crude fiber (3.333 ± 0.37%). Furthermore, the plants inoculated with I. lenis showed the highest concentrations of P (14605 mg/kg), Mg (4964.320 mg/kg), Ca (27389.400 mg/kg), and Mn (86.883 mg/kg). The results of the phytochemical analysis also indicated an increased content of total flavonoids (2.347 mg/g), phenols (3.086 mg/g), tannins (3.902 mg/g), and alkaloids (1.037 mg/g) in the leaf extract of P. frutescens inoculated with I. lenis. Thus, overall the best results of inoculation were observed in Group B i.e. inoculated with I. lenis. GC-MS analysis of methanol leaf extract showed ten bioactive constituents, including 9-Octadecenoic acid (Z)-, methyl ester, and hexadecanoic acid, methyl ester as major constituents found in all the groups of P. frutescens leaves. The phenol (gallic acid) and flavonoids (rutin, kaempferol, and quercetin) were also observed to increase after inoculation by HPTLC analysis. The enhancement in the phytochemical content was co-related with improved anti-oxidant potential which was analyzed by DPPH (% Inhibition: 83.45 µg/ml) and FRAP (2.980 µM Fe (II) equivalent) assay as compared with the control group.</p><p><strong>Conclusion: </strong>Inoculation with I. lenis significantly enhances the uptake of nutritional constituents, phytochemicals, and antioxidant properties in P. frutescens, suggesting its potential to boost the therapeutic properties of host plants.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11316333/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141913292","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 : 2024-08-09DOI: 10.1186/s12934-024-02493-z
Zhao Xingya, Fu Xiaoping, Zhen Jie, Yang Jun, Zheng Hongchen, Bai Wenqin, Song Hui
Background: To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.
Results: Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R-M- without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.
Conclusions: The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restrict
背景:为了有效地将质粒引入芽孢杆菌,并对芽孢杆菌基质菌株进行遗传操作,必须优化转化方法。这些方法旨在延长能力期和提高细胞膜的通透性,以促进外源 DNA 的进入。虽然已经探索了各种策略,但很少有研究深入研究与能力增强相关的代谢物和途径。此外,具有无功能限制性修饰系统的衍生芽孢杆菌菌株在转化外源 DNA 方面表现出更高的效率,但对限制性修饰系统对转化过程的调控缺乏更多的探索:结果:通过转录组比较发现了在斯比前转化条件下,枯草芽孢杆菌 168 中 BsuMI 甲基化修饰组的能力形成机制和调控途径,推测其可能是细胞对碳源的优先选择以及利用碳源时对特定代谢途径的偏好。研究发现,细胞利用糖酵解途径来利用环境中的葡萄糖,同时减少了对该途径中其他磷酸化前体的需求。这些 ATP-底物竞争性代谢途径的削弱使得更多的 ATP 底物在能力形成过程中被分配到信号转导因子 ComP 的自动磷酸化中,从而提高了关键调控蛋白 ComK 的表达水平。ComK 的表达上调了宿主细胞中淀粉和蔗糖负调控因子 SacX 的表达,强化了葡萄糖作为主要碳源的偏好。限制修饰系统中主要蛋白质 BsuMI 的甲基化修饰基团与氧化磷酸化途径中关键酶的功能修饰有关。BsuMI甲基化修饰基团的缺失导致细胞色素氧化酶亚基的表达量减少,从而导致氧化磷酸化途径减弱,促进了细胞的糖酵解速率,进而改善了ATP分子在能力形成中的分布。以构建的无原生限制性修饰系统的枯草芽孢杆菌 168-R-M- 菌株为基础,成功建立了野生型芽孢杆菌菌株的遗传转化平台。与对照组相比,该平台实现了较高的质粒转化效率,显著提高了 63 倍,并增加了芽孢杆菌的普遍性:结论:总结了限制修饰系统功能蛋白 BsuMI 的增强能力形成机制和调控途径,为进一步研究提供了参考。结论:总结了限制性修饰系统功能蛋白 BsuMI 的增强能力形成机制和调控途径,为进一步研究提供了参考,建立了有效的转化平台,克服了野生型芽孢杆菌 DNA 转化的障碍。
{"title":"BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus.","authors":"Zhao Xingya, Fu Xiaoping, Zhen Jie, Yang Jun, Zheng Hongchen, Bai Wenqin, Song Hui","doi":"10.1186/s12934-024-02493-z","DOIUrl":"10.1186/s12934-024-02493-z","url":null,"abstract":"<p><strong>Background: </strong>To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.</p><p><strong>Results: </strong>Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R<sup>-</sup>M<sup>-</sup> without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.</p><p><strong>Conclusions: </strong>The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restrict","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11311917/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141913291","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 : 2024-08-08DOI: 10.1186/s12934-024-02496-w
Minzhi Liu, Sihan Zhou, Yunsong Cao, Keqin Yang, Yao Xiao, Wei Wang
Background: Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity.
Results: At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully.
Conclusions: We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.
{"title":"Characterization of MAP c21873-1 as a new counter-selectable marker for unmarked genetic modification of Pichia pastoris.","authors":"Minzhi Liu, Sihan Zhou, Yunsong Cao, Keqin Yang, Yao Xiao, Wei Wang","doi":"10.1186/s12934-024-02496-w","DOIUrl":"10.1186/s12934-024-02496-w","url":null,"abstract":"<p><strong>Background: </strong>Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity.</p><p><strong>Results: </strong>At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (P<sub>DAS1</sub> and P<sub>FDH1</sub>) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (P<sub>DAS2</sub>) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated P<sub>DAS2</sub> enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully.</p><p><strong>Conclusions: </strong>We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11312372/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141907056","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}
Background: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body.
Results: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group.
Conclusion: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.
{"title":"Lipid A-modified Escherichia coli can produce porcine parvovirus virus-like particles with high immunogenicity and minimal endotoxin activity.","authors":"Xuegang Shen, Yong-Bo Yang, Yanfei Gao, Shujie Wang, Haiwei Wang, Mingxia Sun, Fandan Meng, Yan-Dong Tang, Yabin Tu, Qingke Kong, Tong-Qing An, Xue-Hui Cai","doi":"10.1186/s12934-024-02497-9","DOIUrl":"10.1186/s12934-024-02497-9","url":null,"abstract":"<p><strong>Background: </strong>A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body.</p><p><strong>Results: </strong>In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group.</p><p><strong>Conclusion: </strong>Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11308658/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141907057","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 : 2024-08-08DOI: 10.1186/s12934-024-02498-8
Yurong Xu, Meng Liu, Ruidong Zhao, Yue Pan, Panpan Wu, Chi Zhang, Xiangying Chi, Buchang Zhang, Hang Wu
Background: The TetR family of transcriptional regulators (TFRs), serving as crucial regulators of diverse cellular processes, undergo conformational changes induced by small-molecule ligands, which either inhibit or activate them to modulate target gene expression. Some ligands of TFRs in actinomycetes and their regulatory effects have been identified and studied; however, regulatory mechanisms of the TetR family in the lincomycin-producing Streptomyces lincolnensis remain poorly understood.
Results: In this study, we found that AbrT (SLCG_1979), a TetR family regulator, plays a pivotal role in regulating lincomycin production and morphological development in S. lincolnensis. Deletion of abrT gene resulted in increased lincomycin A (Lin-A) production, but delayed mycelium formation and sporulation on solid media. AbrT directly or indirectly repressed the expression of lincomycin biosynthetic (lin) cluster genes and activated that of the morphological developmental genes amfC, whiB, and ftsZ. We demonstrated that AbrT bound to two motifs (5'-CGCGTACTCGTA-3' and 5'-CGTACGATAGCT-3') present in the bidirectional promoter between abrT and SLCG_1980 genes. This consequently repressed abrT itself and its adjacent gene SLCG_1980 that encodes an arabinose efflux permease. D-arabinose, not naturally occurring as L-arabinose, was identified as the effector molecule of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. Furthermore, based on functional analysis of the AbrT homologue in Saccharopolyspora erythraea, we inferred that the TetR family regulator AbrT may play an important role in regulating secondary metabolism in actinomycetes.
Conclusions: AbrT functions as a regulator for governing lincomycin production and morphological development of S. lincolnensis. Our findings demonstrated that D-arabinose acts as a ligand of AbrT to mediate the regulation of lincomycin biosynthesis in S. lincolnensis. Our findings provide novel insights into ligand-mediated regulation in antibiotic biosynthesis.
{"title":"TetR family regulator AbrT controls lincomycin production and morphological development in Streptomyces lincolnensis.","authors":"Yurong Xu, Meng Liu, Ruidong Zhao, Yue Pan, Panpan Wu, Chi Zhang, Xiangying Chi, Buchang Zhang, Hang Wu","doi":"10.1186/s12934-024-02498-8","DOIUrl":"10.1186/s12934-024-02498-8","url":null,"abstract":"<p><strong>Background: </strong>The TetR family of transcriptional regulators (TFRs), serving as crucial regulators of diverse cellular processes, undergo conformational changes induced by small-molecule ligands, which either inhibit or activate them to modulate target gene expression. Some ligands of TFRs in actinomycetes and their regulatory effects have been identified and studied; however, regulatory mechanisms of the TetR family in the lincomycin-producing Streptomyces lincolnensis remain poorly understood.</p><p><strong>Results: </strong>In this study, we found that AbrT (SLCG_1979), a TetR family regulator, plays a pivotal role in regulating lincomycin production and morphological development in S. lincolnensis. Deletion of abrT gene resulted in increased lincomycin A (Lin-A) production, but delayed mycelium formation and sporulation on solid media. AbrT directly or indirectly repressed the expression of lincomycin biosynthetic (lin) cluster genes and activated that of the morphological developmental genes amfC, whiB, and ftsZ. We demonstrated that AbrT bound to two motifs (5'-CGCGTACTCGTA-3' and 5'-CGTACGATAGCT-3') present in the bidirectional promoter between abrT and SLCG_1980 genes. This consequently repressed abrT itself and its adjacent gene SLCG_1980 that encodes an arabinose efflux permease. D-arabinose, not naturally occurring as L-arabinose, was identified as the effector molecule of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. Furthermore, based on functional analysis of the AbrT homologue in Saccharopolyspora erythraea, we inferred that the TetR family regulator AbrT may play an important role in regulating secondary metabolism in actinomycetes.</p><p><strong>Conclusions: </strong>AbrT functions as a regulator for governing lincomycin production and morphological development of S. lincolnensis. Our findings demonstrated that D-arabinose acts as a ligand of AbrT to mediate the regulation of lincomycin biosynthesis in S. lincolnensis. Our findings provide novel insights into ligand-mediated regulation in antibiotic biosynthesis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11308395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141907058","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}