Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach. Significance Statement Chlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited.

[1]  Xiaodong Deng,et al.  A novel Fe deficiency-responsive element (FeRE) regulates the expression of atx1 in Chlamydomonas reinharditii. , 2007, Plant & cell physiology.

[2]  N. Grishin,et al.  Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates. , 2013, Genes & development.

[3]  A. Valencia,et al.  High-confidence prediction of global interactomes based on genome-wide coevolutionary networks , 2008, Proceedings of the National Academy of Sciences.

[4]  Y. Chisti.  Biodiesel from microalgae. , 2007, Biotechnology advances.

[5]  A. Zeng,et al.  Protein design in systems metabolic engineering for industrial strain development , 2013, Biotechnology journal.

[6]  Guoli Ji,et al.  Bioinformatics Analysis of Alternative Polyadenylation in Green Alga Chlamydomonas reinhardtii Using Transcriptome Sequences from Three Different Sequencing Platforms , 2014, G3: Genes, Genomes, Genetics.

[7]  S. Purton,et al.  Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression , 2014, The Plant journal : for cell and molecular biology.

[8]  M. Goldschmidt-Clermont,et al.  The chloroplast transformation toolbox: selectable markers and marker removal. , 2011, Plant biotechnology journal.

[9]  M. Jonikas,et al.  High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA[W][OPEN] , 2014, Plant Cell.

[10]  S. Merchant,et al.  Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. , 1995, The Plant cell.

[11]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[12]  S. Mayfield,et al.  Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. , 2002, The Plant journal : for cell and molecular biology.

[13]  Chaoguang Tian,et al.  Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation. , 2013, Genomics.

[14]  V. S. Reddy,et al.  Genetic transformation of the green alga: Chlamydomonas reinhardtii by Agrobacterium tumefaciens , 2004 .

[15]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[16]  W. Duckworth,et al.  Recombinant DNA technology in the treatment of diabetes: insulin analogs. , 2001, Endocrine reviews.

[17]  S. Mayfield,et al.  Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. , 2010, Plant biotechnology journal.

[18]  C. Remacle,et al.  The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii , 2009, Eukaryotic Cell.

[19]  A. Brueggeman,et al.  Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii , 2014, Eukaryotic Cell.

[20]  D. González-Halphen,et al.  Identification of Novel Mitochondrial Protein Components ofChlamydomonas reinhardtii. A Proteomic Approach1 , 2003, Plant Physiology.

[21]  Howard J. Li,et al.  Rapid and tunable post-translational coupling of genetic circuits , 2014, Nature.

[22]  Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance , 2011, Journal of Applied Phycology.

[23]  S. Mayfield,et al.  Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization. , 2011, Plant biotechnology journal.

[24]  Michael E Webb,et al.  Thiamine biosynthesis in algae is regulated by riboswitches , 2007, Proceedings of the National Academy of Sciences.

[25]  P. Hegemann,et al.  Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. , 2013, The Plant journal : for cell and molecular biology.

[26]  S. Purton,et al.  Synthesis of Recombinant Products in the Chloroplast , 2014 .

[27]  K. van Dijk,et al.  Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing , 2008, Genetics.

[28]  P. Hegemann,et al.  A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. , 1999, The Plant journal : for cell and molecular biology.

[29]  B. Dujon,et al.  European functional analysis network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome (minireview) , 1998, Electrophoresis.

[30]  Elizabeth H. Harris,et al.  Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation , 2004, Molecular and General Genetics MGG.

[31]  C. Liang,et al.  Genome-Wide Analysis of Tandem Repeats in Plants and Green Algae , 2013, G3: Genes, Genomes, Genetics.

[32]  A. Beyly,et al.  Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii1[W][OPEN] , 2014, Plant Physiology.

[33]  W. Marshall,et al.  Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[34]  W. Jeong,et al.  Stable Plastid Transformation in Nicotiana benthamiana , 2009, Journal of Plant Biology.

[35]  O. Kruse,et al.  Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii. , 2012, Journal of biotechnology.

[36]  M. Schroda,et al.  The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. , 2000, The Plant journal : for cell and molecular biology.

[37]  J. Keasling.  Synthetic biology and the development of tools for metabolic engineering. , 2012, Metabolic engineering.

[38]  S. Purton,et al.  Domestication of the green alga Chlorella sorokiniana: reduction of antenna size improves light-use efficiency in a photobioreactor , 2014, Biotechnology for Biofuels.

[39]  Ovidiu Ruecker,et al.  Strategies to facilitate transgene expression in Chlamydomonas reinhardtii , 2009, Planta.

[40]  M. Hippler,et al.  Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtii , 2006, Proteomics.

[41]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[42]  Sara L. Zimmer,et al.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions , 2007, Science.

[43]  J. Rochaix,et al.  The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii , 2001, Molecular Genetics and Genomics.

[44]  Bas Teusink,et al.  Metabolic shifts: a fitness perspective for microbial cell factories , 2012, Biotechnology Letters.

[45]  John R. Haliburton,et al.  Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. , 2011, Metabolic engineering.

[46]  M. Hippler,et al.  Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii. , 2001, The Plant journal : for cell and molecular biology.

[47]  Daniel Karcher,et al.  Generation of Chlamydomonas strains that efficiently express nuclear transgenes. , 2009, The Plant journal : for cell and molecular biology.

[48]  Richard A Lerner,et al.  Expression and assembly of a fully active antibody in algae , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  K. Hellingwerf,et al.  Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae. , 2013, Current opinion in biotechnology.

[50]  K. Buchholz,et al.  The roots—a short history of industrial microbiology and biotechnology , 2013, Applied Microbiology and Biotechnology.

[51]  P. K. Ajikumar,et al.  The future of metabolic engineering and synthetic biology: towards a systematic practice. , 2012, Metabolic engineering.

[52]  E. H. Harris,et al.  CHLAMYDOMONAS AS A MODEL ORGANISM. , 2003, Annual review of plant physiology and plant molecular biology.

[53]  A. Elazzazy,et al.  Microalgal lipids biochemistry and biotechnological perspectives. , 2014, Biotechnology advances.

[54]  Ursula Goodenough,et al.  Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii , 2009, Eukaryotic Cell.

[55]  J. Nickelsen,et al.  Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. , 1994, The EMBO journal.

[56]  S. Purton,et al.  Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker , 2000, Molecular and General Genetics MGG.

[57]  G. Samuelsson,et al.  Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. , 1997, The Biochemical journal.

[58]  Jeffrey J. Tabor,et al.  Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals , 2014, Nature Methods.

[59]  Wenxu Zhou,et al.  The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion* , 2009, The Journal of Biological Chemistry.

[60]  M. Koornneef,et al.  Naturally occurring genetic variation in Arabidopsis thaliana. , 2004, Annual review of plant biology.

[61]  Juergen E. W. Polle,et al.  tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size , 2003, Planta.

[62]  Lili Xu,et al.  Improvement of hydrogen yield of lba-transgenic Chlamydomonas reinhardtii caused by increasing respiration and impairing photosynthesis , 2014 .

[63]  David Botstein,et al.  Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae , 2014, Nucleic acids research.

[64]  Leo Eberl,et al.  Essence of life: essential genes of minimal genomes. , 2011, Trends in cell biology.

[65]  Ruben G. A. van Heck,et al.  Green genes: bioinformatics and systems-biology innovations drive algal biotechnology. , 2014, Trends in biotechnology.

[66]  Yusuf Chisti,et al.  A matter of detail: assessing the true potential of microalgal biofuels. , 2013, Biotechnology and bioengineering.

[67]  Jay D Keasling,et al.  Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. , 2009, Metabolic engineering.

[68]  M. Schroda,et al.  An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance , 2010, Current Genetics.

[69]  P. May,et al.  An integrative approach towards completing genome-scale metabolic networks. , 2009, Molecular bioSystems.

[70]  P. Falkowski,et al.  Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum. , 2016, The Plant journal : for cell and molecular biology.

[71]  M. Spalding,et al.  TALE activation of endogenous genes in Chlamydomonas reinhardtii , 2014 .

[72]  A. I.,et al.  Neural Field Continuum Limits and the Structure–Function Partitioning of Cognitive–Emotional Brain Networks , 2023, Biology.

[73]  C. Howe,et al.  Biodiesel from algae: challenges and prospects. , 2010, Current opinion in biotechnology.

[74]  Chaoying Yu,et al.  Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols , 2011, Applied Microbiology and Biotechnology.

[75]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[76]  M. Moran,et al.  A brief history. , 2004, Journal of the Medical Association of Georgia.

[77]  J. Rochaix,et al.  The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. , 1989, The EMBO journal.

[78]  M. Goldschmidt-Clermont,et al.  Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. , 1991, Nucleic acids research.

[79]  Jason A. Papin,et al.  Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism , 2011, Molecular systems biology.

[80]  S. Briggs,et al.  Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide , 2012, PloS one.

[81]  Ko Kato,et al.  Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the lac regulation system from Escherichia coli. , 2007, Journal of bioscience and bioengineering.

[82]  A. Grossman,et al.  High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. , 1998, Genetics.

[83]  Y. Li-Beisson,et al.  Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism , 2011, Proteomics.

[84]  P. Maliga.  Plastid Transformation in Flowering Plants , 2012 .

[85]  J. Rochaix,et al.  Selectable marker recycling in the chloroplast , 1996, Molecular and General Genetics MGG.

[86]  J. Keasling,et al.  Engineering dynamic pathway regulation using stress-response promoters , 2013, Nature Biotechnology.

[87]  D. Lewis,et al.  Biofuels from microalgae - an integrated process for a niche fuel market , 2013 .

[88]  Ian K. Blaby,et al.  The Chlamydomonas genome project: a decade on. , 2014, Trends in plant science.

[89]  E. Fernández,et al.  Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. , 2007, Journal of biotechnology.

[90]  Judy Qiu,et al.  Total Synthesis of a Functional Designer Eukaryotic Chromosome , 2014, Science.

[91]  A. Grossman,et al.  Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. , 1992, Nucleic acids research.

[92]  S. Mayfield,et al.  Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes , 2005, Molecular Genetics and Genomics.

[93]  V. Lumbreras,et al.  Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron , 1998 .

[94]  M. Pellegrini,et al.  Transcriptome-Wide Changes in Chlamydomonas reinhardtii Gene Expression Regulated by Carbon Dioxide and the CO2-Concentrating Mechanism Regulator CIA5/CCM1[W][OA] , 2012, Plant Cell.

[95]  Ragnar Tveterås,et al.  A techno-economic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: Research challenges and possibilities , 2015 .

[96]  T M Klein,et al.  Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. , 1988, Science.

[97]  B. Mueller‐Roeber,et al.  Green Transcription Factors: A Chlamydomonas Overview , 2008, Genetics.

[98]  Peter Berthold,et al.  An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. , 2002, Protist.

[99]  Jay D Keasling,et al.  Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. , 2007, Metabolic engineering.

[100]  K. Richards,et al.  Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[101]  David M. Goodstein,et al.  Phytozome: a comparative platform for green plant genomics , 2011, Nucleic Acids Res..

[102]  P. Lefebvre,et al.  Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase , 1989, The Journal of cell biology.

[103]  Rajib Saha,et al.  Synthetic biology of cyanobacteria: unique challenges and opportunities , 2013, Front. Microbiol..

[104]  M. Rahire,et al.  Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops in Chlamydomonas[W] , 2013, Plant Cell.

[105]  J. Napier,et al.  Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids☆ , 2014, Metabolic engineering.

[106]  Ehud Shapiro,et al.  Accurate, Model-Based Tuning of Synthetic Gene Expression Using Introns in S. cerevisiae , 2014, PLoS genetics.

[107]  John Dixon,et al.  Development perspectives of the biobased economy: a review. , 2010 .

[108]  Debashish Bhattacharya,et al.  A molecular timeline for the origin of photosynthetic eukaryotes. , 2004, Molecular biology and evolution.

[109]  S. Sasso,et al.  Unraveling Vitamin B12-Responsive Gene Regulation in Algae1[W] , 2014, Plant Physiology.

[110]  Alison G. Smith,et al.  Do Red and Green Make Brown?: Perspectives on Plastid Acquisitions within Chromalveolates , 2011, Eukaryotic Cell.

[111]  R H Wijffels,et al.  Edible oils from microalgae: insights in TAG accumulation. , 2014, Trends in biotechnology.

[112]  Lake-Ee Quek,et al.  AlgaGEM – a genome-scale metabolic reconstruction of algae based on the Chlamydomonas reinhardtii genome , 2011, BMC Genomics.

[113]  Thomas F. Knight,et al.  Idempotent Vector Design for Standard Assembly of Biobricks , 2003 .

[114]  Manuel Porcar,et al.  iGEM 2.0—refoundations for engineering biology , 2014, Nature Biotechnology.

[115]  S. Mayfield,et al.  Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses , 2014, Photosynthesis Research.

[116]  T. Zhao,et al.  Gene Silencing by Artificial Micrornas in Chlamydomonas , 2008 .

[117]  Mark F. Rogers,et al.  Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii , 2010, BMC Genomics.

[118]  J. Collins,et al.  A brief history of synthetic biology , 2014, Nature Reviews Microbiology.

[119]  Ovidiu Ruecker,et al.  Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii , 2008, Molecular Genetics and Genomics.

[120]  Gary D Bader,et al.  Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants , 2001, Science.

[121]  K. Kindle.  High-frequency nuclear transformation of Chlamydomonas reinhardtii. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[122]  Alison G. Smith,et al.  Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity , 2013, Proceedings of the National Academy of Sciences.

[123]  J. Rochaix,et al.  Characterisation of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation , 1995 .

[124]  S. Merchant,et al.  TAG, you're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. , 2012, Current opinion in biotechnology.

[125]  C. Howe,et al.  Triacylglyceride Production and Autophagous Responses in Chlamydomonas reinhardtii Depend on Resource Allocation and Carbon Source , 2014, Eukaryotic Cell.

[126]  H. Koop,et al.  Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum , 1993, Bio/Technology.

[127]  Ralph Bock,et al.  Synthetic biology in plastids. , 2014, The Plant journal : for cell and molecular biology.

[128]  Julian N. Rosenberg,et al.  Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. , 2013, The Plant journal : for cell and molecular biology.

[129]  Keiko Ikeda,et al.  Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter , 2014, Plant biotechnology journal.

[130]  P. May,et al.  ChlamyCyc: an integrative systems biology database and web-portal for Chlamydomonas reinhardtii , 2009, BMC Genomics.

[131]  J. Alric,et al.  Central Carbon Metabolism and Electron Transport in Chlamydomonas reinhardtii: Metabolic Constraints for Carbon Partitioning between Oil and Starch , 2013, Eukaryotic Cell.

[132]  Robert Verpoorte,et al.  Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering , 2011, Applied Microbiology and Biotechnology.

[133]  J. Yates,et al.  Proteomic Analysis of Isolated Chlamydomonas Centrioles Reveals Orthologs of Ciliary-Disease Genes , 2005, Current Biology.

[134]  R. Quatrano.  Genomics , 1998, Plant Cell.

[135]  R. Loppes,et al.  Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii , 1997, Current Genetics.

[136]  C. Dussap,et al.  A model‐based method for investigating bioenergetic processes in autotrophically growing eukaryotic microalgae: Application to the green algae Chlamydomonas reinhardtii , 2011, Biotechnology progress.

[137]  D. Bhattacharya,et al.  Using Natural Selection to Explore the Adaptive Potential of Chlamydomonas reinhardtii , 2014, PloS one.

[138]  Karen M Polizzi.  What is synthetic biology? , 2013, Methods in molecular biology.

[139]  J. Rochaix,et al.  Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. , 2011, Plant biotechnology journal.

[140]  L Bogorad,et al.  Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome. , 1989, The Plant cell.

[141]  Matteo Pellegrini,et al.  The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii , 2014, Eukaryotic Cell.

[142]  Matthew D. Wilkerson,et al.  PlantGDB: a resource for comparative plant genomics , 2007, Nucleic Acids Res..

[143]  Jason A. Papin,et al.  Metabolic network analysis integrated with transcript verification for sequenced genomes , 2009, Nature Methods.

[144]  Quanxi Wang,et al.  Improved biohydrogen production with an expression of codon-optimized hemH and lba genes in the chloroplast of Chlamydomonas reinhardtii. , 2011, Bioresource technology.

[145]  Andrew R. Bassett,et al.  Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. , 2009, The Plant journal : for cell and molecular biology.

[146]  W. Fang.  Transcriptome-Wide Changes in Chlamydomonas reinhardtii Gene Expression Regulated by Carbon Dioxide and the CO 2 -Concentrating Mechanism , 2012 .

[147]  C. Tomlin,et al.  Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay , 2013, Nucleic acids research.

[148]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[149]  H. Neumann,et al.  Synthetic biology approaches in drug discovery and pharmaceutical biotechnology , 2010, Applied Microbiology and Biotechnology.

[150]  V. Danilenko,et al.  Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker. , 1996, Gene.

[151]  Miller Tran,et al.  Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. , 2007, Plant biotechnology journal.