Metabolic Engineering Communications最新文献_第4页

Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis 利用连接子序列多样性融合胡萝卜素环化酶和羟化酶合成玉米黄质

IF 5.2

Metabolic Engineering Communications Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00222

Aurélie Bouin , Congqiang Zhang , Nic D. Lindley , Gilles Truan , Thomas Lautier

{"title":"Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis","authors":"Aurélie Bouin , Congqiang Zhang , Nic D. Lindley , Gilles Truan , Thomas Lautier","doi":"10.1016/j.mec.2023.e00222","DOIUrl":"10.1016/j.mec.2023.e00222","url":null,"abstract":"<div>Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and β-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bc/34/main.PMC10165439.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9452847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS) 枯草芽孢杆菌的适应性实验室进化以克服蒸馏器干燥谷物中木质纤维素水解物的毒性（DDGS）

IF 5.2

Metabolic Engineering Communications Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00223

Jasper L.S.P. Driessen , Josefin Johnsen , Ivan Pogrebnyakov , Elsayed T.T. Mohamed , Solange I. Mussatto , Adam M. Feist , Sheila I. Jensen , Alex T. Nielsen

{"title":"Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS)","authors":"Jasper L.S.P. Driessen , Josefin Johnsen , Ivan Pogrebnyakov , Elsayed T.T. Mohamed , Solange I. Mussatto , Adam M. Feist , Sheila I. Jensen , Alex T. Nielsen","doi":"10.1016/j.mec.2023.e00223","DOIUrl":"https://doi.org/10.1016/j.mec.2023.e00223","url":null,"abstract":"<div>Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of Bacillus subtilis strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator codY in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (katA, perR) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50170514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering sorghum for higher 4-hydroxybenzoic acid content 工程高粱具有较高的4-羟基苯甲酸含量

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00207

Chien-Yuan Lin , Yang Tian , Kimberly Nelson-Vasilchik , Joel Hague , Ramu Kakumanu , Mi Yeon Lee , Venkataramana R. Pidatala , Jessica Trinh , Christopher M. De Ben , Jutta Dalton , Trent R. Northen , Edward E.K. Baidoo , Blake A. Simmons , John M. Gladden , Corinne D. Scown , Daniel H. Putnam , Albert P. Kausch , Henrik V. Scheller , Aymerick Eudes

{"title":"Engineering sorghum for higher 4-hydroxybenzoic acid content","authors":"Chien-Yuan Lin , Yang Tian , Kimberly Nelson-Vasilchik , Joel Hague , Ramu Kakumanu , Mi Yeon Lee , Venkataramana R. Pidatala , Jessica Trinh , Christopher M. De Ben , Jutta Dalton , Trent R. Northen , Edward E.K. Baidoo , Blake A. Simmons , John M. Gladden , Corinne D. Scown , Daniel H. Putnam , Albert P. Kausch , Henrik V. Scheller , Aymerick Eudes","doi":"10.1016/j.mec.2022.e00207","DOIUrl":"10.1016/j.mec.2022.e00207","url":null,"abstract":"<div>Engineering bioenergy crops to accumulate coproducts in planta can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (ubiC) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-d-arabino-heptulonate-7-phosphate synthase (aroG) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express ubiC and aroG produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11–15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9519784/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40393831","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Engineering of Yarrowia lipolytica for terpenoid production 产萜类化合物的聚脂耶氏菌工程

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00213

Jonathan Asmund Arnesen, Irina Borodina

引用次数: 6

A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway 一种用于细胞外和细胞内代谢物定量的靶向代谢组学方法，涵盖了完整的单脂素和木脂素合成途径

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00205

Andrea Steinmann , Katrin Schullehner , Anna Kohl , Christina Dickmeis , Maurice Finger , Georg Hubmann , Guido Jach , Ulrich Commandeur , Marco Girhard , Vlada B. Urlacher , Stephan Lütz

{"title":"A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway","authors":"Andrea Steinmann , Katrin Schullehner , Anna Kohl , Christina Dickmeis , Maurice Finger , Georg Hubmann , Guido Jach , Ulrich Commandeur , Marco Girhard , Vlada B. Urlacher , Stephan Lütz","doi":"10.1016/j.mec.2022.e00205","DOIUrl":"10.1016/j.mec.2022.e00205","url":null,"abstract":"<div>Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization.In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites.The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9474286/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40365479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production 调整一个高性能的多重crispri恶臭假单胞菌菌株，进一步提高靛蓝苷的产量

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00206

Jeffrey J. Czajka , Deepanwita Banerjee , Thomas Eng , Javier Menasalvas , Chunsheng Yan , Nathalie Munoz Munoz , Brenton C. Poirier , Young-Mo Kim , Scott E. Baker , Yinjie J. Tang , Aindrila Mukhopadhyay

{"title":"Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production","authors":"Jeffrey J. Czajka , Deepanwita Banerjee , Thomas Eng , Javier Menasalvas , Chunsheng Yan , Nathalie Munoz Munoz , Brenton C. Poirier , Young-Mo Kim , Scott E. Baker , Yinjie J. Tang , Aindrila Mukhopadhyay","doi":"10.1016/j.mec.2022.e00206","DOIUrl":"10.1016/j.mec.2022.e00206","url":null,"abstract":"<div>In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6–fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2–fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5–fold and 1.8–fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/15/32/main.PMC9494242.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33482926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate 对香豆酸酯生产β-酮己二酸假单胞菌KT2440的crispr干扰工具集的动态和单细胞特性

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00204

Jacob A. Fenster , Allison Z. Werner , Jian Wei Tay , Matthew Gillen , Leo Schirokauer , Nicholas C. Hill , Audrey Watson , Kelsey J. Ramirez , Christopher W. Johnson , Gregg T. Beckham , Jeffrey C. Cameron , Carrie A. Eckert

{"title":"Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate","authors":"Jacob A. Fenster , Allison Z. Werner , Jian Wei Tay , Matthew Gillen , Leo Schirokauer , Nicholas C. Hill , Audrey Watson , Kelsey J. Ramirez , Christopher W. Johnson , Gregg T. Beckham , Jeffrey C. Cameron , Carrie A. Eckert","doi":"10.1016/j.mec.2022.e00204","DOIUrl":"10.1016/j.mec.2022.e00204","url":null,"abstract":"<div>Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9460563/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33461891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus 假地曲霉衣康酸的产生受LaeA调控

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00203

Kyle R. Pomraning , Ziyu Dai , Nathalie Munoz , Young-Mo Kim , Yuqian Gao , Shuang Deng , Teresa Lemmon , Marie S. Swita , Jeremy D. Zucker , Joonhoon Kim , Stephen J. Mondo , Ellen Panisko , Meagan C. Burnet , Bobbie-Jo M. Webb-Robertson , Beth Hofstad , Scott E. Baker , Kristin E. Burnum-Johnson , Jon K. Magnuson , for the Agile BioFoundry

{"title":"Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus","authors":"Kyle R. Pomraning , Ziyu Dai , Nathalie Munoz , Young-Mo Kim , Yuqian Gao , Shuang Deng , Teresa Lemmon , Marie S. Swita , Jeremy D. Zucker , Joonhoon Kim , Stephen J. Mondo , Ellen Panisko , Meagan C. Burnet , Bobbie-Jo M. Webb-Robertson , Beth Hofstad , Scott E. Baker , Kristin E. Burnum-Johnson , Jon K. Magnuson , for the Agile BioFoundry","doi":"10.1016/j.mec.2022.e00203","DOIUrl":"10.1016/j.mec.2022.e00203","url":null,"abstract":"<div>The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4e/ad/main.PMC9440423.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40352282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides 专用金门组装平台(RtGGA)的开发

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00200

Nemailla Bonturi , Marina Julio Pinheiro , Paola Monteiro de Oliveira , Eka Rusadze , Tobias Eichinger , Gintare Liudžiūtė , Juliano Sabedotti De Biaggi , Age Brauer , Maido Remm , Everson Alves Miranda , Rodrigo Ledesma-Amaro , Petri-Jaan Lahtvee

{"title":"Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides","authors":"Nemailla Bonturi , Marina Julio Pinheiro , Paola Monteiro de Oliveira , Eka Rusadze , Tobias Eichinger , Gintare Liudžiūtė , Juliano Sabedotti De Biaggi , Age Brauer , Maido Remm , Everson Alves Miranda , Rodrigo Ledesma-Amaro , Petri-Jaan Lahtvee","doi":"10.1016/j.mec.2022.e00200","DOIUrl":"10.1016/j.mec.2022.e00200","url":null,"abstract":"<div>Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000098/pdfft?md5=a2fe424d543e5912a0eacd9cd6daa4d6&pid=1-s2.0-S2214030122000098-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46506584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus RNA聚合酶ii驱动的CRISPR-Cas9系统高效非生长偏倚克卢维酵母代谢工程

IF 5.2

Metabolic Engineering Communications Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00208

Danielle Bever , Ian Wheeldon , Nancy Da Silva

{"title":"RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus","authors":"Danielle Bever , Ian Wheeldon , Nancy Da Silva","doi":"10.1016/j.mec.2022.e00208","DOIUrl":"https://doi.org/10.1016/j.mec.2022.e00208","url":null,"abstract":"<div>The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.</div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000177/pdfft?md5=f6be45d36815b9de356945d06bf05598&pid=1-s2.0-S2214030122000177-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91591437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2