Synthetic and Systems Biotechnology最新文献_第2页

Coupling genome-wide continuous perturbation with biosensor screening reveals the potential targets in yeast isopentanol synthesis network

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-30 DOI: 10.1016/j.synbio.2024.12.010

Qi Xiao , Jingjing Shi , Lixian Wang , Guoping Zhao , Yanfei Zhang

The increasing consumption of fossil fuels is contributing to global resource depletion and environmental pollution. Branched-chain higher alcohols, such as isopentanol and isobutanol, have attracted significant attention as next-generation biofuels. Biofuel production through microbial fermentation offers a green, sustainable, and renewable alternative to chemical synthesis. While enhanced production of isopentanol has been achieved in a variety of chassis, the fermentation yield has not yet reached levels suitable for industrial-scale production. In this study, we employed a continuous perturbation tool to construct a genome-scale perturbation library, combined with an isopentanol biosensor to screen for high-yielding mutants. We identified five high-yielding mutants, each exhibiting an increased glucose conversion rate and isopentanol titer. The F2 strain, in particular, achieved an isopentanol titer of 1.57 ± 0.014 g/L and a yield of 14.04 ± 0.251 mg/g glucose (10% glucose), surpassing the highest values reported to date in engineered Saccharomyces cerevisiae. Systematic transcriptome analysis of the isopentanol synthesis, glycolysis, glycerol metabolism, and ethanol synthesis pathways revealed that MPC, OAC1, BAT2, GUT2, PDC6, and ALD4 are linked to efficient isopentanol production. Further analysis of differentially expressed genes (DEGs) identified 17 and 12 co-expressed DEGs (co-DEGs) in all mutants and the two second-round mutants, respectively. In addition, we validated the knockout or overexpression of key co-DEGs. Our results confirmed the critical roles of HOM3 and DIP5 in isopentanol production, along with genes associated with the aerobic respiratory chain (SDH3, CYT1, COX7, ROX1, and ATG41) and cofactor balance (BNA2 and NDE1). Additionally, functional analysis of the co-DEGs revealed that MAL33 is associated with the synthesis of branched-chain higher alcohols, expanding the intracellular metabolic network and offering new possibilities for green, cost-effective biofuel production.

{"title":"Coupling genome-wide continuous perturbation with biosensor screening reveals the potential targets in yeast isopentanol synthesis network","authors":"Qi Xiao , Jingjing Shi , Lixian Wang , Guoping Zhao , Yanfei Zhang","doi":"10.1016/j.synbio.2024.12.010","DOIUrl":"10.1016/j.synbio.2024.12.010","url":null,"abstract":"<div><div>The increasing consumption of fossil fuels is contributing to global resource depletion and environmental pollution. Branched-chain higher alcohols, such as isopentanol and isobutanol, have attracted significant attention as next-generation biofuels. Biofuel production through microbial fermentation offers a green, sustainable, and renewable alternative to chemical synthesis. While enhanced production of isopentanol has been achieved in a variety of chassis, the fermentation yield has not yet reached levels suitable for industrial-scale production. In this study, we employed a continuous perturbation tool to construct a genome-scale perturbation library, combined with an isopentanol biosensor to screen for high-yielding mutants. We identified five high-yielding mutants, each exhibiting an increased glucose conversion rate and isopentanol titer. The F2 strain, in particular, achieved an isopentanol titer of 1.57 ± 0.014 g/L and a yield of 14.04 ± 0.251 mg/g glucose (10% glucose), surpassing the highest values reported to date in engineered <em>Saccharomyces cerevisiae</em>. Systematic transcriptome analysis of the isopentanol synthesis, glycolysis, glycerol metabolism, and ethanol synthesis pathways revealed that <em>MPC</em>, <em>OAC1</em>, <em>BAT2</em>, <em>GUT2</em>, <em>PDC6</em>, and <em>ALD4</em> are linked to efficient isopentanol production. Further analysis of differentially expressed genes (DEGs) identified 17 and 12 co-expressed DEGs (co-DEGs) in all mutants and the two second-round mutants, respectively. In addition, we validated the knockout or overexpression of key co-DEGs. Our results confirmed the critical roles of <em>HOM3</em> and <em>DIP5</em> in isopentanol production, along with genes associated with the aerobic respiratory chain (<em>SDH3</em>, <em>CYT1</em>, <em>COX7</em>, <em>ROX1</em>, and <em>ATG41</em>) and cofactor balance (<em>BNA2</em> and <em>NDE1</em>). Additionally, functional analysis of the co-DEGs revealed that <em>MAL33</em> is associated with the synthesis of branched-chain higher alcohols, expanding the intracellular metabolic network and offering new possibilities for green, cost-effective biofuel production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 452-462"},"PeriodicalIF":4.4,"publicationDate":"2024-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143161058","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}

引用次数: 0

Design and structure of overlapping regions in PCA via deep learning

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-27 DOI: 10.1016/j.synbio.2024.12.007

Yan Zheng , Xi-Chen Cui , Fei Guo , Ming-Liang Dou , Ze-Xiong Xie , Ying-Jin Yuan

Polymerase cycling assembly (PCA) stands out as the predominant method in the synthesis of kilobase-length DNA fragments. The design of overlapping regions is the core factor affecting the success rate of synthesis. However, there still exists DNA sequences that are challenging to design and construct in the genome synthesis. Here we proposed a deep learning model based on extensive synthesis data to discern latent sequence representations in overlapping regions with an AUPR of 0.805. Utilizing the model, we developed the SmartCut algorithm aimed at designing oligonucleotides and enhancing the success rate of PCA experiments. This algorithm was successfully applied to sequences with diverse synthesis constraints, 80.4 % of which were synthesized in a single round. We further discovered structure differences represented by major groove width, stagger, slide, and centroid distance between overlapping and non-overlapping regions, which elucidated the model's reasonableness through the lens of physical chemistry. This comprehensive approach facilitates streamlined and efficient investigations into the genome synthesis.

{"title":"Design and structure of overlapping regions in PCA via deep learning","authors":"Yan Zheng , Xi-Chen Cui , Fei Guo , Ming-Liang Dou , Ze-Xiong Xie , Ying-Jin Yuan","doi":"10.1016/j.synbio.2024.12.007","DOIUrl":"10.1016/j.synbio.2024.12.007","url":null,"abstract":"<div><div>Polymerase cycling assembly (PCA) stands out as the predominant method in the synthesis of kilobase-length DNA fragments. The design of overlapping regions is the core factor affecting the success rate of synthesis. However, there still exists DNA sequences that are challenging to design and construct in the genome synthesis. Here we proposed a deep learning model based on extensive synthesis data to discern latent sequence representations in overlapping regions with an AUPR of 0.805. Utilizing the model, we developed the SmartCut algorithm aimed at designing oligonucleotides and enhancing the success rate of PCA experiments. This algorithm was successfully applied to sequences with diverse synthesis constraints, 80.4 % of which were synthesized in a single round. We further discovered structure differences represented by major groove width, stagger, slide, and centroid distance between overlapping and non-overlapping regions, which elucidated the model's reasonableness through the lens of physical chemistry. This comprehensive approach facilitates streamlined and efficient investigations into the genome synthesis.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 442-451"},"PeriodicalIF":4.4,"publicationDate":"2024-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143161542","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}

引用次数: 0

Genetically-modified activation strategy facilitates the discovery of sesquiterpene-derived metabolites from Penicillium brasilianum 转基因激活策略促进了巴西青霉倍半萜衍生代谢物的发现。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-25 DOI: 10.1016/j.synbio.2024.12.006

Wenni He , Xiaoting Rong , Hui Lv , Lihua Zhang , Jinglin Bai , Lu Wang , Liyan Yu , Lixin Zhang , Tao Zhang

Genome mining has revealed that Penicillium spp. possess numerous down-regulated or cryptic biosynthetic gene clusters (BGCs). This finding hinted that our investigation of fungal secondary metabolomes is limited. Herein, we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by Penicillium brasilianum CGMCC 3.4402. The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene berA responsible for berkeleyacetals biosynthesis from Neosartorya glabra. Chemical analysis of the extracts from the mutant strain Pb-OE:berA enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C (1), one daucane-type arpenicarotane C (4), along with four known sesquiterpenoids including arpenibisabolane A (2), eupenicisirenins A (3), arpenicarotane B (5) and aspterric acid (6). The assignments of their structures were elucidated from detailed analyses of spectroscopic data, electronic circular dichroism calculation, and biogenetic considerations. The bioassay of isolated compounds (1–6) exhibited no cytotoxic activities against three tumor cells including MCF-7, HepG2, and A549. Arpenibisabolane C (1) and A (2) showed weak inhibition bioactivities on aquatic pathogens Vibrio owensii and Vibrio algivorus. Moreover, phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed. Based on the chemical structures and biogenetic investigations, a hypothetic pathway of new compounds (1, 4) was proposed.

基因组挖掘揭示了青霉菌具有许多下调或隐藏的生物合成基因簇（BGCs）。这一发现提示我们对真菌次生代谢组的研究是有限的。在此，我们报道了一种转基因激活策略来表征巴西青霉CGMCC 3.4402产生的倍半萜类化合物的光谱。新树胶缩醛生物合成通路特异性调控基因berA的组成表达刺激了隐化或下调的通路。对突变菌株Pb-OE:berA的提取物进行化学分析，分离出2个新化合物，包括1个双abolene型arpenibisabolane C(1)、1个daucane型arpenisarotane C(4)，以及4个已知的倍半萜类化合物，包括arpenibisabolane A(2)、eupenicisirenins A(3)、arpenisarotane B(5)和aspasparacid(6)。通过详细的光谱分析、电子圆二色性计算，对它们的结构进行了鉴定。还有生物遗传学方面的考虑。分离得到的化合物（1-6）对MCF-7、HepG2和A549三种肿瘤细胞无细胞毒活性。青蒿素C(1)和A(2)对水生病原菌乌氏弧菌和藻弧菌的抑制活性较弱。此外，还进行了系统发育分析和关键倍半萜合成酶的序列比对。基于化学结构和生物遗传学研究，提出了新化合物（1,4）的合成途径。

{"title":"Genetically-modified activation strategy facilitates the discovery of sesquiterpene-derived metabolites from Penicillium brasilianum","authors":"Wenni He , Xiaoting Rong , Hui Lv , Lihua Zhang , Jinglin Bai , Lu Wang , Liyan Yu , Lixin Zhang , Tao Zhang","doi":"10.1016/j.synbio.2024.12.006","DOIUrl":"10.1016/j.synbio.2024.12.006","url":null,"abstract":"<div><div>Genome mining has revealed that <em>Penicillium</em> spp. possess numerous down-regulated or cryptic biosynthetic gene clusters (BGCs). This finding hinted that our investigation of fungal secondary metabolomes is limited. Herein, we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by <em>Penicillium brasilianum</em> CGMCC 3.4402. The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene <em>berA</em> responsible for berkeleyacetals biosynthesis from <em>Neosartorya glabra</em>. Chemical analysis of the extracts from the mutant strain <em>Pb</em>-OE:<em>berA</em> enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C (<strong>1</strong>), one daucane-type arpenicarotane C (<strong>4</strong>), along with four known sesquiterpenoids including arpenibisabolane A (<strong>2</strong>), eupenicisirenins A (<strong>3</strong>), arpenicarotane B (<strong>5</strong>) and aspterric acid (<strong>6</strong>). The assignments of their structures were elucidated from detailed analyses of spectroscopic data, electronic circular dichroism calculation, and biogenetic considerations. The bioassay of isolated compounds (<strong>1</strong>–<strong>6</strong>) exhibited no cytotoxic activities against three tumor cells including MCF-7, HepG2, and A549. Arpenibisabolane C (<strong>1</strong>) and A (<strong>2</strong>) showed weak inhibition bioactivities on aquatic pathogens <em>Vibrio owensii</em> and <em>Vibrio algivorus</em>. Moreover, phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed. Based on the chemical structures and biogenetic investigations, a hypothetic pathway of new compounds (<strong>1</strong>, <strong>4</strong>) was proposed.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 391-400"},"PeriodicalIF":4.4,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11745945/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143011963","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}

引用次数: 0

Metabolic engineering of Glarea lozoyensis for high-level production of pneumocandin B0 高水平产气肺菌素B0的聚氮草代谢工程研究。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-24 DOI: 10.1016/j.synbio.2024.12.008

Xinyi Zhang , Shu Cheng , Jing Yang , Li Lu , Zixin Deng , Guangkai Bian , Tiangang Liu

Pneumocandin B₀ (PB₀) is a lipohexapeptide synthesized by Glarea lozoyensis and serves as the precursor for the widely used antifungal drug caspofungin acetate (Cancidas®). However, the low titer of PB₀ results in fermentation and purification costs during caspofungin production, limiting its widespread clinical application. Here, we engineered an efficient PB₀-producing strain of G. lozoyensis by systems metabolic engineering strategies, including multi-omics analysis and multilevel metabolic engineering. We overexpressed four rate-limiting enzymes: thioesterase GLHYD, two cytochrome P450s GLP450s, and chorismate synthase GLCS; knocked out two competing pathways responsible for producing 6-methylsalicylic acid and pyranidine E; and overexpressed the global transcriptional activator GLHYP. As a result, the PB₀ titer increased by 108.7 % to 2.63 g/L at the shake-flask level through combinatorial strategies. Our study provides valuable insights into achieving high-level production of PB₀ and offers general guidance for developing efficient fungal cell factories to produce polyketide synthase-non-ribosomal peptide synthetase hybrid metabolites.

肺结核菌素B0 （PB0）是一种由Glarea lozoyensis合成的脂己肽，是广泛使用的抗真菌药物caspofungin acetate （Cancidas®）的前体。然而，低滴度的PB0导致了caspofunins生产过程中的发酵和纯化成本，限制了其广泛的临床应用。本研究通过多组学分析和多层次代谢工程等系统代谢工程策略，构建了高效产pb0的lozoyensis菌株。我们过表达了四种限速酶：硫酯酶GLHYD，两种细胞色素p4500s， glp4500s和choris酸合成酶GLCS；敲除了负责产生6-甲基水杨酸和吡啶E的两条相互竞争的途径；并过度表达全局转录激活因子GLHYP。结果表明，通过组合策略，摇瓶水平PB0滴度提高了108.7%，达到2.63 g/L。我们的研究为实现PB0的高水平生产提供了有价值的见解，并为开发高效的真菌细胞工厂生产聚酮合成酶-非核糖体肽合成酶杂交代谢物提供了一般指导。

{"title":"Metabolic engineering of Glarea lozoyensis for high-level production of pneumocandin B0","authors":"Xinyi Zhang , Shu Cheng , Jing Yang , Li Lu , Zixin Deng , Guangkai Bian , Tiangang Liu","doi":"10.1016/j.synbio.2024.12.008","DOIUrl":"10.1016/j.synbio.2024.12.008","url":null,"abstract":"<div><div>Pneumocandin B<sub>0</sub> (PB<sub>0</sub>) is a lipohexapeptide synthesized by <em>Glarea lozoyensis</em> and serves as the precursor for the widely used antifungal drug caspofungin acetate (Cancidas®). However, the low titer of PB<sub>0</sub> results in fermentation and purification costs during caspofungin production, limiting its widespread clinical application. Here, we engineered an efficient PB<sub>0</sub>-producing strain of <em>G. lozoyensis</em> by systems metabolic engineering strategies, including multi-omics analysis and multilevel metabolic engineering. We overexpressed four rate-limiting enzymes: thioesterase GLHYD, two cytochrome P450s GLP450s, and chorismate synthase GLCS; knocked out two competing pathways responsible for producing 6-methylsalicylic acid and pyranidine E; and overexpressed the global transcriptional activator GLHYP. As a result, the PB<sub>0</sub> titer increased by 108.7 % to 2.63 g/L at the shake-flask level through combinatorial strategies. Our study provides valuable insights into achieving high-level production of PB<sub>0</sub> and offers general guidance for developing efficient fungal cell factories to produce polyketide synthase-non-ribosomal peptide synthetase hybrid metabolites.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 381-390"},"PeriodicalIF":4.4,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742615/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143011966","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}

引用次数: 0

Environment signal dependent biocontainment systems for engineered organisms: Leveraging triggered responses and combinatorial systems 工程生物体的环境信号依赖生物控制系统：利用触发反应和组合系统。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-20 DOI: 10.1016/j.synbio.2024.12.005

Shreya Varma , Khushi Ash Gulati , Janani Sriramakrishnan , Riyaa Kedar Ganla , Ritu Raval

As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers—chemical, light, temperature, and pH—this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

随着合成生物学的进步，为基因工程生物（geo）制定强有力的生物控制战略的必要性越来越重要，以减轻与其潜在的环境释放相关的生物安全风险。本文旨在评估工程生物的环境信号依赖生物控制系统，特别关注利用触发反应和组合系统。有不同类型的触发器-化学，光，温度和ph -这篇综述说明了如何设计这些系统来响应环境信号，确保更高的安全性。它还侧重于组合生物防护，以避免GEO意外释放到外部环境中的后果。讨论了案例研究，以演示这些系统在实际场景中的实际应用。

引用次数: 0

Characterization of Aspergillus oryzae mutant and its application in heterologous lipase expression 米曲霉突变体的鉴定及其在外源脂肪酶表达中的应用。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-20 DOI: 10.1016/j.synbio.2024.12.003

Qinghua Li , Chen Zhang , Jianghua Li , Guocheng Du , Zhaofeng Li , Jingwen Zhou , Guoqiang Zhang

The Aspergillus oryzae expression system has been developed into a chassis for the production of heterologous lipases, attributed to its strong capabilities in protein production and secretion, robust post-translational modifications, and favourable safety profile. However, the system's relatively low expression levels remain a challenge, hindering its ability to meet the increasing demands of large-scale production. Strain C19, screened by high-throughput methods combining droplet microfluidics and flow cytometry, was demonstrated to be a potential chassis cell based on fermentation kinetic analysis and transcriptome sequencing. By leveraging the endogenous α-amylase's expression elements and integration sites, a combination of random and site-directed integration strategies was employed to enhance the expression of heterologous lipases in strain C19. As a result, lipase production in shake-flask fermentation reached a titer of 113.6 U/L. The study further demonstrated that the different α-amylase gene loci could serve as effective integration sites for the multi-copy expression of heterologous proteins because the lipase activity of the 3-amylase site integrated strain C19#1-ABC was 3.3 times higher than that of C19#1. Furthermore, fermentation results in a 5-L bioreactor indicated that optimization of fermentation processes and facilities had the potential to further increase heterologous protein expression levels. These findings offered valuable insights into the advancement of A. oryzae expression systems and the potential for scaling engineered strains for industrial applications.

米曲霉表达系统由于其强大的蛋白质生产和分泌能力、强大的翻译后修饰和良好的安全性，已发展成为生产外源脂肪酶的基础。然而，该系统相对较低的表达水平仍然是一个挑战，阻碍了其满足大规模生产日益增长的需求的能力。菌株C19采用微滴微流体和流式细胞术相结合的高通量筛选方法，通过发酵动力学分析和转录组测序证实为潜在的底盘细胞。利用内源性α-淀粉酶的表达元件和整合位点，采用随机整合和位点定向整合相结合的策略增强了外源脂肪酶在菌株C19中的表达。结果表明，摇瓶发酵脂肪酶的效价达到113.6 U/L。研究进一步表明，不同α-淀粉酶基因位点可以作为外源蛋白多拷贝表达的有效整合位点，因为3-淀粉酶位点整合菌株C19#1- abc的脂肪酶活性比C19#1高3.3倍。此外，在5-L生物反应器中的发酵结果表明，优化发酵工艺和设施有可能进一步提高外源蛋白的表达水平。这些发现为米芽孢杆菌表达系统的进步和规模化工程菌株的工业应用潜力提供了有价值的见解。

{"title":"Characterization of Aspergillus oryzae mutant and its application in heterologous lipase expression","authors":"Qinghua Li , Chen Zhang , Jianghua Li , Guocheng Du , Zhaofeng Li , Jingwen Zhou , Guoqiang Zhang","doi":"10.1016/j.synbio.2024.12.003","DOIUrl":"10.1016/j.synbio.2024.12.003","url":null,"abstract":"<div><div>The <em>Aspergillus oryzae</em> expression system has been developed into a chassis for the production of heterologous lipases, attributed to its strong capabilities in protein production and secretion, robust post-translational modifications, and favourable safety profile. However, the system's relatively low expression levels remain a challenge, hindering its ability to meet the increasing demands of large-scale production. Strain C19, screened by high-throughput methods combining droplet microfluidics and flow cytometry, was demonstrated to be a potential chassis cell based on fermentation kinetic analysis and transcriptome sequencing. By leveraging the endogenous α-amylase's expression elements and integration sites, a combination of random and site-directed integration strategies was employed to enhance the expression of heterologous lipases in strain C19. As a result, lipase production in shake-flask fermentation reached a titer of 113.6 U/L. The study further demonstrated that the different α-amylase gene loci could serve as effective integration sites for the multi-copy expression of heterologous proteins because the lipase activity of the 3-amylase site integrated strain C19#1-ABC was 3.3 times higher than that of C19#1. Furthermore, fermentation results in a 5-L bioreactor indicated that optimization of fermentation processes and facilities had the potential to further increase heterologous protein expression levels. These findings offered valuable insights into the advancement of <em>A. oryzae</em> expression systems and the potential for scaling engineered strains for industrial applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 365-372"},"PeriodicalIF":4.4,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742560/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143011903","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}

引用次数: 0

Enhancing abscisic acid production in Botrytis cinerea through metabolic engineering based on a constitutive promoter library 基于组成启动子文库的代谢工程提高灰葡萄孢脱落酸产量。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-20 DOI: 10.1016/j.synbio.2024.12.004

Ling-Ru Wang , Ji-Zi-Hao Tang , Shu-Ting Zhu , Na Wu , Zhi-Kui Nie , Tian-Qiong Shi

Abscisic acid (ABA) is an important plant growth regulator with broad applications in agriculture, forestry, and other fields. Currently, the industrial production of ABA primarily relies on microbial fermentation using Botrytis cinerea, but its genetic toolbox is limited. To address this, we first screened 10 strong constitutive promoters from the genome of B. cinerea through transcriptomic analysis. The expression levels of the promoters covered a range of 3–4 orders of magnitude according to the measured β-glucuronidase activity. Subsequently, four promoters of different strength were used to balance the cofactor supply in B. cinerea. Overexpression of NADH kinase using the medium-strength promoter Pef1a significantly enhanced ABA production, resulting in a 32.26 % increase compared to the control. Finally, by combining promoter engineering with a push-pull strategy, we optimized the biosynthesis of ABA. The recombinant strain Pthi4:hmgr-Pef1a:a4, overexpressing HMGR under the Pthi4 promoter and Bcaba4 under the Pef1a promoter, achieved an ABA titer of 1.18 g/L, a 58.92 % increase. To our best knowledge, this is the first constitutive promoter library suitable for B. cinerea, providing important tools for the industrial production of ABA.

脱落酸是一种重要的植物生长调节剂，在农业、林业等领域有着广泛的应用。目前，ABA的工业生产主要依赖于利用灰霉病菌（Botrytis cinerea）进行微生物发酵，但其遗传工具箱有限。为了解决这个问题，我们首先通过转录组学分析从灰孢杆菌基因组中筛选了10个强组成启动子。根据测定的β-葡萄糖醛酸酶活性，启动子的表达水平覆盖了3-4个数量级的范围。随后，使用4种不同强度的启动子来平衡绿僵菌的辅因子供应。使用中等强度启动子Pef1a过表达NADH激酶显著提高了ABA的产生，与对照相比增加了32.26%。最后，我们将启动子工程与推拉策略相结合，优化了ABA的生物合成。重组菌株Pthi4: hgr -Pef1a:a4在Pthi4启动子下过表达HMGR，在Pef1a启动子下过表达Bcaba4，获得了1.18 g/L的ABA效价，提高了58.92%。据我们所知，这是第一个适合于灰孢菌的组成启动子文库，为ABA的工业化生产提供了重要的工具。

{"title":"Enhancing abscisic acid production in Botrytis cinerea through metabolic engineering based on a constitutive promoter library","authors":"Ling-Ru Wang , Ji-Zi-Hao Tang , Shu-Ting Zhu , Na Wu , Zhi-Kui Nie , Tian-Qiong Shi","doi":"10.1016/j.synbio.2024.12.004","DOIUrl":"10.1016/j.synbio.2024.12.004","url":null,"abstract":"<div><div>Abscisic acid (ABA) is an important plant growth regulator with broad applications in agriculture, forestry, and other fields. Currently, the industrial production of ABA primarily relies on microbial fermentation using <em>Botrytis cinerea</em>, but its genetic toolbox is limited. To address this, we first screened 10 strong constitutive promoters from the genome of <em>B. cinerea</em> through transcriptomic analysis. The expression levels of the promoters covered a range of 3–4 orders of magnitude according to the measured β-glucuronidase activity. Subsequently, four promoters of different strength were used to balance the cofactor supply in <em>B. cinerea</em>. Overexpression of NADH kinase using the medium-strength promoter <em>Pef1a</em> significantly enhanced ABA production, resulting in a 32.26 % increase compared to the control. Finally, by combining promoter engineering with a push-pull strategy, we optimized the biosynthesis of ABA. The recombinant strain Pthi4:hmgr-Pef1a:a4, overexpressing HMGR under the <em>Pthi4</em> promoter and Bcaba4 under the <em>Pef1a</em> promoter, achieved an ABA titer of 1.18 g/L, a 58.92 % increase. To our best knowledge, this is the first constitutive promoter library suitable for <em>B. cinerea</em>, providing important tools for the industrial production of ABA.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 373-380"},"PeriodicalIF":4.4,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11742572/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143011959","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}

引用次数: 0

Modeling coding sequence design for virus-based expression in tobacco 为基于病毒的烟草表达设计编码序列模型。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-11 DOI: 10.1016/j.synbio.2024.12.002

Moritz Burghardt , Tamir Tuller

Transient expression in Tobacco is a popular way to produce recombinant proteins in plants. The design of various expression vectors, delivered into the plant by Agrobacterium, has enabled high production levels of some proteins. To further enhance expression, researchers often adapt the coding sequence of heterologous genes to the host, but this strategy has produced mixed results in Tobacco.

To study the effects of different sequence features on protein yield, we compile a dataset of the yields and coding sequences of previously published expression studies of more than 200 coding sequences.

We evaluate various established gene expression models on a subset of the expression studies. We find that use of tobacco codons is only moderately predictive of protein yield as informative sequence features likely extend over multiple codons. Additionally, we show that codon usage of organisms that use tobacco as a host for expression of their proteins in a similar way as the synthetic system, like viruses and agrobacteria, can be used to predict heterologous expression. Other predictive features are related to tRNA supply and demand, the inclusion of a translational ramp of codons with lower adaptation to the tRNA pool at the beginning of the coding region, and the amino acid composition of the recombinant protein. A model based on all the features achieved a correlation of 0.57 with protein yield.

We believe that our study provides a practical guideline for coding sequence design for efficient expression in tobacco.

烟草瞬时表达是一种在植物中产生重组蛋白的常用方法。设计各种表达载体，通过农杆菌传递到植物中，使一些蛋白质的高产量成为可能。为了进一步增强表达，研究人员经常将异源基因的编码序列适应宿主，但这种策略在烟草中产生了不同的结果。为了研究不同序列特征对蛋白产率的影响，我们编制了一个数据集，其中包含了200多个已发表的表达研究的编码序列的产率和编码序列。我们在表达研究的一个子集上评估各种已建立的基因表达模型。我们发现烟草密码子的使用只能适度预测蛋白质产量，因为信息序列特征可能延伸到多个密码子上。此外，我们还发现，以烟草为宿主表达其蛋白质的生物，如病毒和农杆菌，密码子的使用方式与合成系统类似，可用于预测异源表达。其他预测特征与tRNA的供需、编码区开头对tRNA库适应性较低的密码子翻译斜坡的包含以及重组蛋白的氨基酸组成有关。基于所有特征的模型与蛋白质产量的相关系数为0.57。我们相信我们的研究为烟草高效表达的编码序列设计提供了实用的指导。

{"title":"Modeling coding sequence design for virus-based expression in tobacco","authors":"Moritz Burghardt , Tamir Tuller","doi":"10.1016/j.synbio.2024.12.002","DOIUrl":"10.1016/j.synbio.2024.12.002","url":null,"abstract":"<div><div>Transient expression in Tobacco is a popular way to produce recombinant proteins in plants. The design of various expression vectors, delivered into the plant by <em>Agrobacterium</em>, has enabled high production levels of some proteins. To further enhance expression, researchers often adapt the coding sequence of heterologous genes to the host, but this strategy has produced mixed results in Tobacco.</div><div>To study the effects of different sequence features on protein yield, we compile a dataset of the yields and coding sequences of previously published expression studies of more than 200 coding sequences.</div><div>We evaluate various established gene expression models on a subset of the expression studies. We find that use of tobacco codons is only moderately predictive of protein yield as informative sequence features likely extend over multiple codons. Additionally, we show that codon usage of organisms that use tobacco as a host for expression of their proteins in a similar way as the synthetic system, like viruses and agrobacteria, can be used to predict heterologous expression. Other predictive features are related to tRNA supply and demand, the inclusion of a translational ramp of codons with lower adaptation to the tRNA pool at the beginning of the coding region, and the amino acid composition of the recombinant protein. A model based on all the features achieved a correlation of 0.57 with protein yield.</div><div>We believe that our study provides a practical guideline for coding sequence design for efficient expression in tobacco.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 337-345"},"PeriodicalIF":4.4,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11718241/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142971954","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}

引用次数: 0

Construction of antibiotic-free riboflavin producer in Escherichia coli by metabolic engineering strategies with a plasmid stabilization system 利用质粒稳定系统的代谢工程策略构建大肠杆菌无抗生素核黄素生产体。

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-12-06 DOI: 10.1016/j.synbio.2024.12.001

Xiaoling Zhang , Yanan Li , Kang Wang , Jilong Yin , Yuxuan Du , Zhen Yang , Xuewei Pan , Jiajia You , Zhiming Rao

Riboflavin, an important vitamin utilized in pharmaceutical products and as a feed additive, is mainly produced by metabolically engineered bacterial fermentation. However, the reliance on antibiotics in the production process leads to increased costs and safety risks. To address these challenges, an antibiotic-free Escherichia coli riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system. Initially, competitive pathways and feedback inhibition were attenuated to enhance the metabolic flux towards riboflavin. Key genes in the purine pathway were overexpressed to boost the availability of riboflavin precursors. Subsequently, a plasmid stabilization system based on toxin was screened and characterized, achieving a plasmid retention rate of 84.9% after 10 days of passaging. Finally, transcriptomic analysis at the genome-wide level revealed several rate-limiting genes, including pgl, gnd, and yigB, which were subsequently upregulated, leading to a 26% improvement in riboflavin production. With optimization of the culture medium, the final strain allowed the production of 11.5 g/L of riboflavin with a yield of 90.4 mg/g glucose in 5 L bioreactors without antibiotics. These strategies can be extended to other plasmid-based riboflavin derivative production systems.

核黄素是一种重要的维生素，用于医药产品和饲料添加剂，主要是通过代谢工程细菌发酵生产的。然而，在生产过程中对抗生素的依赖导致了成本和安全风险的增加。为了解决这些挑战，利用代谢工程方法结合一种新的质粒稳定系统构建了一种无抗生素的大肠杆菌核黄素生产者。最初，竞争途径和反馈抑制被减弱，以增强对核黄素的代谢通量。嘌呤途径中的关键基因被过度表达以提高核黄素前体的可用性。随后，筛选并鉴定了基于毒素的质粒稳定体系，传代10天后质粒保留率为84.9%。最后，全基因组水平的转录组学分析揭示了几个限速基因，包括pgl、gnd和yigB，这些基因随后被上调，导致核黄素产量提高26%。通过对培养基的优化，最终菌株在无抗生素的5 L生物反应器中，核黄素产量为11.5 g/L，葡萄糖产量为90.4 mg/g。这些策略可以扩展到其他基于质粒的核黄素衍生物生产系统。

{"title":"Construction of antibiotic-free riboflavin producer in Escherichia coli by metabolic engineering strategies with a plasmid stabilization system","authors":"Xiaoling Zhang , Yanan Li , Kang Wang , Jilong Yin , Yuxuan Du , Zhen Yang , Xuewei Pan , Jiajia You , Zhiming Rao","doi":"10.1016/j.synbio.2024.12.001","DOIUrl":"10.1016/j.synbio.2024.12.001","url":null,"abstract":"<div><div>Riboflavin, an important vitamin utilized in pharmaceutical products and as a feed additive, is mainly produced by metabolically engineered bacterial fermentation. However, the reliance on antibiotics in the production process leads to increased costs and safety risks. To address these challenges, an antibiotic-free <em>Escherichia coli</em> riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system. Initially, competitive pathways and feedback inhibition were attenuated to enhance the metabolic flux towards riboflavin. Key genes in the purine pathway were overexpressed to boost the availability of riboflavin precursors. Subsequently, a plasmid stabilization system based on toxin was screened and characterized, achieving a plasmid retention rate of 84.9% after 10 days of passaging. Finally, transcriptomic analysis at the genome-wide level revealed several rate-limiting genes, including <em>pgl</em>, <em>gnd</em>, and <em>yigB</em>, which were subsequently upregulated, leading to a 26% improvement in riboflavin production. With optimization of the culture medium, the final strain allowed the production of 11.5 g/L of riboflavin with a yield of 90.4 mg/g glucose in 5 L bioreactors without antibiotics. These strategies can be extended to other plasmid-based riboflavin derivative production systems.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 2","pages":"Pages 346-355"},"PeriodicalIF":4.4,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11731478/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984877","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}

引用次数: 0

Semi-rational design and modification of phosphoketolase to improve the yield of tyrosol in Saccharomyces cerevisiae 半合理设计和修饰磷酸酮醇酶以提高酿酒酵母酪醇的产率

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-11-26 DOI: 10.1016/j.synbio.2024.11.007

Na Song , Huili Xia , Yaoru Xie , Shuaikang Guo , Rong Zhou , Lingling Shangguan , Kun Zhuang , Huiyan Zhang , Feiran An , Xueyun Zheng , Lan Yao , Shihui Yang , Xiong Chen , Jun Dai

Tyrosol is an important component of pharmaceuticals, nutraceuticals, and cosmetics, and their biosynthetic pathways are currently a hot research topic. d-Erythrose 4-phosphate is a key precursor for the biosynthesis of tyrosol in Saccharomyces cerevisiae. Hence, the flux of d-Erythrose 4-phosphate determined the yield of tyrosol synthesis. In this study, we first obtained an S. cerevisiae strain S19 with a tyrosol yield of 247.66 mg/L by metabolic engineering strategy. To increase the production of d-Erythrose 4-phosphate, highly active phosphoketolase BA-C was obtained by bioinformatics combined with tyrosol yield assay. The key residue sites 183, 217, and 320 were obtained by molecular docking, kinetic simulation, and tyrosol yield verification. After mutation, the highly efficient phosphoketolase BA-C^His320Met was obtained, with a 37.32 % increase in enzyme activity. The tyrosol production of strain S26 with BA-C^His320Arg increased by 43.05 % than strain S25 with BA-C and increased by 151.19 % compared with the strain S19 without phosphoketolase in a 20 L fermenter. The mining and modification of phosphoketolase will provide strong support for the de novo synthesis of aromatic compounds.

Tyrosol是药品、保健品和化妆品的重要成分，其生物合成途径是目前研究的热点。d- 4-磷酸红酶是酿酒酵母生物合成酪醇的关键前体。因此，d- 4-磷酸红酶的通量决定了酪醇合成的产率。在本研究中，我们首先通过代谢工程策略获得了一株酿酒葡萄球菌S19，其酪醇产量为247.66 mg/L。为了提高d- 4-磷酸红酶的产量，生物信息学结合酪醇产率测定获得了高活性的磷酸酮醇酶BA-C。通过分子对接、动力学模拟和酪醇产率验证得到了关键残基位点183、217和320。突变后获得了高效磷酸酮酶BA-CHis320Met，酶活性提高了37.32%。在20 L发酵罐中，添加BA-CHis320Arg的菌株S26的酪醇产量比添加BA-C的菌株S25提高了43.05%，比不添加磷酸酮醇酶的菌株S19提高了151.19%。磷酸酮醇酶的挖掘和改性将为芳香族化合物的新合成提供有力的支持。

{"title":"Semi-rational design and modification of phosphoketolase to improve the yield of tyrosol in Saccharomyces cerevisiae","authors":"Na Song , Huili Xia , Yaoru Xie , Shuaikang Guo , Rong Zhou , Lingling Shangguan , Kun Zhuang , Huiyan Zhang , Feiran An , Xueyun Zheng , Lan Yao , Shihui Yang , Xiong Chen , Jun Dai","doi":"10.1016/j.synbio.2024.11.007","DOIUrl":"10.1016/j.synbio.2024.11.007","url":null,"abstract":"<div><div>Tyrosol is an important component of pharmaceuticals, nutraceuticals, and cosmetics, and their biosynthetic pathways are currently a hot research topic. <span>d</span>-Erythrose 4-phosphate is a key precursor for the biosynthesis of tyrosol in <em>Saccharomyces cerevisiae</em>. Hence, the flux of <span>d</span>-Erythrose 4-phosphate determined the yield of tyrosol synthesis. In this study, we first obtained an <em>S. cerevisiae</em> strain S19 with a tyrosol yield of 247.66 mg/L by metabolic engineering strategy. To increase the production of <span>d</span>-Erythrose 4-phosphate, highly active phosphoketolase BA-C was obtained by bioinformatics combined with tyrosol yield assay. The key residue sites 183, 217, and 320 were obtained by molecular docking, kinetic simulation, and tyrosol yield verification. After mutation, the highly efficient phosphoketolase BA-C<sup>His320Met</sup> was obtained, with a 37.32 % increase in enzyme activity. The tyrosol production of strain S26 with BA-C<sup>His320Arg</sup> increased by 43.05 % than strain S25 with BA-C and increased by 151.19 % compared with the strain S19 without phosphoketolase in a 20 L fermenter. The mining and modification of phosphoketolase will provide strong support for the de novo synthesis of aromatic compounds.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"10 1","pages":"Pages 294-306"},"PeriodicalIF":4.4,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142745796","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}

引用次数: 0