Article
Biochemical Research Methods
Yingjia Tong, Jingwen Zhou, Liang Zhang, Peng Xu
Summary: Violacein, a naturally occurring anticancer therapeutic compound, was produced in Y. lipolytica using a library of strains constructed with Golden Gate assembly method. Gene expression analysis revealed that strong expression of VioB, VioC, and VioD favored violacein production, while weak expression of VioD was associated with high deoxyviolacein production. By optimizing culture conditions, violacein reached a maximum of 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks.
ACS SYNTHETIC BIOLOGY
(2021)
Article
Biotechnology & Applied Microbiology
Ya-Wen Li, Cai-Ling Yang, Qi Shen, Qian-Qian Peng, Qi Guo, Zhi-Kui Nie, Xiao-Man Sun, Tian-Qiong Shi, Xiao-Jun Ji, He Huang
Summary: In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Yarrowia lipolytica. By optimizing key factors, the assembly efficiency of multiple fragments reached up to 90%. The system was successfully applied for the overproduction of (-)-alpha-bisabolol, achieving the highest titer reported in yeast to date. This study expands the toolbox of metabolic engineering and enables efficient production of various terpenoids.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Biochemical Research Methods
Vijaydev Ganesan, Lummy Monteiro, Dheeraj Pedada, Anthony Stohr, Mark Blenner
Summary: Yarrowia lipolytica is an industrial host with a high fatty acid flux. In this study, we developed a rapid multiplexed strain engineering tool using base editors for efficient gene editing. The base editors were found to be less toxic than CRISPR-Cas9 for multiplexed gene editing. By optimizing the editing conditions and using a co-selection approach, we achieved high-efficiency multiplexed gene editing.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Yongshuo Ma, Jingbo Li, Sanwen Huang, Gregory Stephanopoulos
Summary: This study explores the strategy of expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica to achieve efficient synthesis of astaxanthin, leading to increased yield and reduced accumulation of intermediates. Targeting the astaxanthin pathway to subcellular organelles accelerates conversion and significantly improves synthesis, ultimately enhancing compartmentalized isoprenoid biosynthesis in Y. lipolytica.
METABOLIC ENGINEERING
(2021)
Article
Biotechnology & Applied Microbiology
Dan-Ni Wang, Jie Feng, Chen-Xi Yu, Xin-Kai Zhang, Jun Chen, Liu-Jing Wei, Zhijie Liu, Liming Ouyang, Lixin Zhang, Qiang Hua, Feng Liu
Summary: This study successfully synthesized high-value astaxanthin using genetically engineered yeast. By optimizing enzyme expression and addressing key bottlenecks in the biosynthesis process, the production of astaxanthin was significantly increased. The study also analyzed the impact of astaxanthin biosynthesis on cell metabolism, providing important insights for the production of high-value carotenoids.
SYNTHETIC AND SYSTEMS BIOTECHNOLOGY
(2022)
Article
Agriculture, Multidisciplinary
Jing Chen, Ruiling Zhang, Guilin Zhang, Zhen Liu, Hong Jiang, Xiangzhao Mao
Summary: In this study, glycosylated astaxanthin was successfully produced in yeast for the first time. Through modular assembly and expression regulation, the yield of astaxanthin was increased by 4.9-fold. Finally, introduction of the crtX gene resulted in the highest reported yield of microbially produced glycosylated astaxanthin (1.47 mg/L).
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
(2023)
Article
Biochemistry & Molecular Biology
Zhiqian Wang, Yunjun Yan, Houjin Zhang
Summary: In this study, a light-induced expression system was developed in Yarrowia lipolytica for the synthesis and verification of Bleomycin resistance protein (BleoR). The system demonstrated potential for transcriptional regulation of target genes and construction of large-scale synthetic networks.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Microbiology
Shiyu Liu, Yao Sun, Tianhui Wei, Dianliang Gong, Qi Wang, Zhe Zhan, Jinzhu Song
Summary: This study constructed a recombinant strain and discovered the degradation pathway of 3-HP in Y. lipolytica, successfully producing 3-HP in this strain. The yield of 3-HP reached 1.128 g/L in shake flask fermentation and 16.23 g/L in fed-batch fermentation, which are highly competitive compared to other yeast chassis cells. This study lays the foundation for the production of 3-HP in Y. lipolytica and provides a reference for future research.
Article
Biotechnology & Applied Microbiology
Carmen Lopez, Mingfeng Cao, Zhanyi Yao, Zengyi Shao
Summary: The study highlights the importance of plasmid stability for gene expression and the relevance of cargo dependency in plasmid stability. It reveals the unique structure of ARSs from Yarrowia lipolytica, with a previously unannotated spacer playing a critical role in plasmid behavior.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2021)
Article
Biotechnology & Applied Microbiology
Evgeniya Y. Yuzbasheva, Pasquale Scarcia, Tigran Yuzbashev, Eugenia Messina, Iuliia M. Kosikhina, Luigi Palmieri, Artem Shutov, Maria O. Taratynova, Rodrigo Ledesma Amaro, Ferdinando Palmieri, Sergey P. Sineoky, Gennaro Agrimi
Summary: The mitochondrial succinate-fumarate carrier YlSfc1 controls the efflux of isocitric acid from mitochondria in Yarrowia lipolytica, with its overexpression leading to increased production of isocitric acid. Coordinating the expression of YlSFC1 and YlAMPD genes, along with inactivating the YlYHM2 gene, enhances the accumulation of isocitric acid. This study demonstrates the potential of manipulating mitochondrial transport systems to improve product accumulation.
METABOLIC ENGINEERING
(2021)
Article
Biotechnology & Applied Microbiology
Qi Hang Chen, Ya Dan Qian, Yong Jie Niu, Ching Yuan Hu, Yong Hong Meng
Summary: A highly efficient CRISPR-iCas9 genetic manipulation tool was established for Y. lipolytica, enabling rapid gene knockout and one-step integration of large gene fragments. This technology significantly improved the target product yield and offers a feasible approach to genetic modification for efficient biosynthesis.
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Zhiyong Cui, Ziwei Zhu, Jinhong Zhang, Zhennan Jiang, Yinghang Liu, Qian Wang, Jin Hou, Qingsheng Qi
Summary: 5-ALA is a key precursor in the biosynthesis of various important compounds. In this study, engineered strains of Yarrowia lipolytica were used to produce 5-ALA efficiently. By optimizing culture conditions, the titer of 5-ALA reached 2216.8 mg/L in fed-batch fermentation, the highest achieved using yeast globally. This study demonstrates the potential of using SDH-deficient Y. lipolytica as a platform for efficient 5-ALA production.
BIOCHEMICAL ENGINEERING JOURNAL
(2021)
Article
Biochemical Research Methods
Tao Sun, Yizi Yu, Lexin Wang, Yichun Qi, Tian Xu, Zhe Wang, Lu Lin, Rodrigo Ledesma-Amaro, Xiao-Jun Ji
Summary: The production of biodiesel from renewable lignocellulosic biomass using microbial lipids is a promising strategy to promote green energy transition. In this study, an engineered Yarrowia lipolytica strain was constructed using metabolic engineering and adaptive laboratory evolution, which efficiently produced lipids from both glucose and xylose. By adopting the Push-Pull-Block strategy, the lipid content reached 73.42% of the dry cell weight. Furthermore, the engineered strain integrated a heterologous xylose utilization pathway and underwent ALE, resulting in a strain that could accumulate 53.64% DCW of lipids from xylose with a lipid titer of 16.25 g/L. This work highlights the potential of microbial lipid overproduction from lignocellulose using engineered Y. lipolytica.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Nicolas Daffern, Irene M. Francino-Urdaniz, Zachary T. Baumer, Timothy A. Whitehead
Summary: A standardized Golden Gate method for building user-defined libraries is presented, allowing the construction of large, deeply mutagenized protein encoding libraries. This method is efficient and capable of generating libraries with a high number of protein encoding variants. It can be applied to different protein engineering systems and provides general-use destination vectors and detailed protocols for other researchers.
BIOTECHNOLOGY AND BIOENGINEERING
(2023)
Article
Agriculture, Multidisciplinary
Baixi Zhang, Lijuan Ni, Xiaoshu Tang, Xuemei Chen, Bo Hu
Summary: This study achieved a high yield of t10, c12-CLA through genetic modification and reduced its degradation rate. The highest t10, c12-CLA yield reported to date was obtained in a bioreactor using low-cost carbon sources.
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
(2022)
Editorial Material
Biotechnology & Applied Microbiology
Lu Lin, Rodrigo Ledesma-Amaro, Xiao-Jun Ji, He Huang
Summary: Nylons, important polyamide materials, can be synthesized from various monomers. Bio-based nylon monomers are traditionally obtained through chemical conversion from vegetable oils, but they can be more sustainably obtained through multienzymatic catalysis. For large-scale application of this process, enzyme engineering and process innovation must be combined.
TRENDS IN BIOTECHNOLOGY
(2023)
Article
Microbiology
Jaroslav Hambalko, Peter Gajdos, Jean-Marc Nicaud, Rodrigo Ledesma-Amaro, Michal Tupec, Iva Pichova, Milan Certik
Summary: Research has found that sex pheromone mixtures of certain insect species can produce unique C16 long conjugated fatty acids with two or three conjugated double bonds. The supplementation of fatty acid methyl esters into the medium resulted in the identification of 20 new fatty acids with two or three double bonds. The study proves that Y. lipolytica is capable of synthesizing C16-conjugated fatty acids, and further optimization could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.
Article
Biochemistry & Molecular Biology
Lara Selles Vidal, Mark Isalan, John T. Heap, Rodrigo Ledesma-Amaro
Summary: Directed evolution is a powerful tool for protein engineering, allowing for the rapid selection of biomolecule variants with desired properties. Since its initial development in 1967, various techniques have been developed to facilitate genetic diversification and variant isolation. This review discusses the advantages, drawbacks, and recent developments in directed evolution methodologies, highlighting the ability to handle larger library sizes and address new research questions.
RSC CHEMICAL BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Patrycja Szczepanska, Magdalena Rychlicka, Sylwia Groborz, Angelika Kruszynska, Rodrigo Ledesma-Amaro, Andrzej Rapak, Anna Gliszczynska, Zbigniew Lazar
Summary: By lipophilizing RES with fatty acids, several esters were obtained and evaluated for their anticancer and antioxidant properties against different types of cancer cells, as well as their antioxidant effects on normal cells. The RES derivatives showed enhanced biological activity and potential applications in cancer prevention and treatment, as well as oxidative stress suppression.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2023)
Article
Biochemistry & Molecular Biology
Lucas Coppens, Tanya Tschirhart, Dagmar H. Leary, Sophie M. Colston, Jaimee R. Compton, William Judson Hervey, Karl L. Dana, Gary J. Vora, Sergio Bordel, Rodrigo Ledesma-Amaro
Summary: This study presents the first genome-scale metabolic model (GSMM) of V. natriegens, which was validated and compared with empirical data. The model was used for a metabolic comparison between V. natriegens and Escherichia coli, leading to the discovery of the model architecture of V. natriegens' respiratory and ATP-generating system as well as a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data also provided insights into the halophilic adaptations of V. natriegens. Additionally, a Resource Balance Analysis model was created using the GSMM to study carbon resource allocation.
MOLECULAR SYSTEMS BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Georgio Nemer, Nicolas Louka, Paul Rabiller Blandin, Richard G. Maroun, Eugene Vorobiev, Tristan Rossignol, Jean-Marc Nicaud, Erwann Guenin, Mohamed Koubaa
Summary: This study focuses on the biosynthesis of a mixture of violacein and deoxyviolacein using a genetically modified Y. lipolytica strain as a production chassis. The intracellular pigments were extracted and purified using column chromatography. The optimal separation between the pigments was achieved using an ethyl acetate/cyclohexane mixture with different ratios, followed by analysis using thin-layer chromatography and nuclear magnetic resonance.
Article
Biochemistry & Molecular Biology
Simran Kaur Aulakh, Lara Selles Vidal, Eric J. South, Huadong Peng, Sreejith Jayasree Varma, Lucia Herrera-Dominguez, Markus Ralser, Rodrigo Ledesma-Amaro
Summary: In this study, by systematically testing pairwise combinations of auxotrophic Saccharomyces cerevisiae deletion mutants, we identified 49 pairs that spontaneously form syntrophic, synergistic communities. We characterized the stability and growth dynamics of nine cocultures and demonstrated that a pair of tryptophan auxotrophs grow by exchanging a pathway intermediate rather than end products. Additionally, we introduced a malonic semialdehyde biosynthesis pathway split between different pairs of auxotrophs, resulting in increased production. Our findings highlight the potential of nutritional codependence to improve biotechnological processes.
NATURE CHEMICAL BIOLOGY
(2023)
Review
Multidisciplinary Sciences
Alicia E. Graham, Rodrigo Ledesma-Amaro
Summary: This Review article explores the potential of microorganisms as a solution to the challenges faced by our food system. Engineered microorganisms can sustainably produce enhanced foods and ingredients. The authors discuss the technical, economical, and societal limitations, as well as the current and future perspectives.
NATURE COMMUNICATIONS
(2023)
Article
Biochemical Research Methods
Huadong Peng, Ruiqi Chen, William M. Shaw, Piotr Hapeta, Wei Jiang, David J. Bell, Tom Ellis, Rodrigo Ledesma-Amaro
Summary: Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. This study utilized synthetic biology and modular approaches to produce a valuable fragrance compound, raspberry ketone (RK), in the model yeast Saccharomyces cerevisiae. The strategies employed included modular cloning, modular pathway engineering, and modular cocultures, resulting in the production of high levels of RK and its precursor. This research highlights the importance and potential of modularity in synthetic biology for industrial applications.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Tao Sun, Yizi Yu, Lexin Wang, Yichun Qi, Tian Xu, Zhe Wang, Lu Lin, Rodrigo Ledesma-Amaro, Xiao-Jun Ji
Summary: The production of biodiesel from renewable lignocellulosic biomass using microbial lipids is a promising strategy to promote green energy transition. In this study, an engineered Yarrowia lipolytica strain was constructed using metabolic engineering and adaptive laboratory evolution, which efficiently produced lipids from both glucose and xylose. By adopting the Push-Pull-Block strategy, the lipid content reached 73.42% of the dry cell weight. Furthermore, the engineered strain integrated a heterologous xylose utilization pathway and underwent ALE, resulting in a strain that could accumulate 53.64% DCW of lipids from xylose with a lipid titer of 16.25 g/L. This work highlights the potential of microbial lipid overproduction from lignocellulose using engineered Y. lipolytica.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Xiuxia Liu, Manman Sun, Alex Xiong Gao, Rodrigo Ledesma-Amaro, Qiuwu Fang, Yankun Yang, Zhonghu Bai
Summary: In this study, a bicistronic expression cassette based on a leaderless promoter was developed in Corynebacterium glutamicum, which improved the orthogonality and regulatory range of gene expression. The importance of the fore-cistron and SD motif in maintaining the strength of leaderless bicistronic designs was also explored.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Multidisciplinary Sciences
Ting Zhang, Qingdong Zeng, Fan Ji, Honghong Wu, Rodrigo Ledesma-Amaro, Qingshan Wei, Hao Yang, Xuhan Xia, Yao Ren, Keqing Mu, Qiang He, Zhensheng Kang, Ruijie Deng
Summary: An in-field molecular diagnostic tool for crop diseases is developed, which utilizes a cheap colorimetric paper and a smartphone for rapid, low-cost, and multiplexed detection of pathogens. The tool can guide the precise application of pesticides, resulting in reduced pesticide usage and improved crop yield.
NATURE COMMUNICATIONS
(2023)
Article
Biology
Tigran V. Yuzbashev, Evgeniya Y. Yuzbasheva, Olga E. Melkina, Davina Patel, Dmitrii Bubnov, Heiko Dietz, Rodrigo Ledesma-Amaro
Summary: CRISPR/Cas9 technology is revolutionizing microbial cell engineering by enabling marker-free chromosomal integration of DNA. In this study, improvements were made to current Cas9 toolkits to make CRISPR technologies more accessible and easy to implement. These improvements include the ability to switch between marker-free and marker-based integration constructs, redirect multigene integration cassettes to alternative genomic loci, and assemble guide RNA sequences rapidly in vivo. These methodologies were combined with well-established technologies to create a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. The YaliCraft toolkit for Yarrowia lipolytica was developed as a proof of concept, demonstrating the successful integration of markerless DNA and easy assembly of new gRNAs, resulting in the production of homogentisic acid.
COMMUNICATIONS BIOLOGY
(2023)
Article
Chemistry, Multidisciplinary
Xueqin Lv, Shixiu Cui, Jie Chen, Lingrui Wang, Yanfeng Liu, Jianghua Li, Guocheng Du, Xiaohao Liu, Jian Chen, Rodrigo Ledesma-Amaro, Long Liu
Summary: Food proteins are crucial nutrients in our diet, but their acquisition from the agroindustry leads to environmental pollution and high resource consumption. In this study, a chemo-biocascade catalysis (CBCC) system was developed to convert CO2 into lacto-proteins. The results demonstrate the potential of tandem chemocatalysis and biocatalysis for sustainable protein manufacturing.
Article
Biochemical Research Methods
Alicia Climent-Catala, Ivan Casas-Rodrigo, Suhasini Iyer, Rodrigo Ledesma-Amaro, Thomas E. Ouldridge
Summary: This study investigates the performance of RNA light-up aptamers as transcriptional fluorescent reporters and compares them to protein-based reporters. The researchers found that RNA light-up aptamers exhibit suitable characteristics as transcriptional reporters over time and at the single-cell level, with higher variability in a population compared to protein-based reporters. Additionally, these RNA aptamers may offer faster dynamics compared to fluorescent proteins in E. coli. The implementation of these transcriptional reporters can facilitate transcription-based studies and expand the use of RNA-based circuits in bacterial cells.
ACS SYNTHETIC BIOLOGY
(2023)
