Improvement of light to biomass conversion by de-regulation of light-harvesting protein translation in Chlamydomonas reinhardtii.

The efficient use of microalgae to convert sun light energy into biomass is limited by losses during high light illumination of dense cell cultures in closed bioreactors. Uneven light distribution can be overcome by using cell cultures with smaller antenna sizes packed to high cell density cultures, thus allowing good light penetration into the inner sections of the reactor. We engineered a new small PSII antenna size Chlamydomonas reinhardtii strain with improved photon conversion efficiency and increased growth rates under high light conditions. We achieved this goal by transformation of a permanently active variant NAB1* of the LHC translation repressor NAB1 to reduce antenna size via translation repression. NAB1* expression was demonstrated in Stm6Glc4T7 (T7), leading to a reduction of LHC antenna size by 10-17%. T7 showed a approximately 50% increase of photosynthetic efficiency (PhiPSII) at saturating light intensity compared to the parental strain. T7 converted light to biomass with much higher efficiencies with a approximately 50% improved mid log growth phase. Moreover, T7 cultures reached higher densities when grown in large-scale bioreactors. Thus, the phenotype of strain T7 may have important implications for biotechnological applications in which photosynthetic microalgae are used for large-scale culturing as an alternative plant biomass source.

[1]  A. Grossman,et al.  A genome’s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii , 2004, Current Genetics.

[2]  J. Barber,et al.  Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii*S , 2006, Molecular & Cellular Proteomics.

[3]  N. Sueoka,et al.  Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardi. I. Isotopic transfer experiments with a strain producing eight zoospores. , 1967, Journal of molecular biology.

[4]  C. Schwarz,et al.  Translational control of photosynthetic gene expression in phototrophic eukaryotes. , 2008, Physiologia plantarum.

[5]  P. Lefebvre,et al.  The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation , 1994, Molecular and cellular biology.

[6]  P. Falkowski,et al.  Chloroplast redox regulation of nuclear gene transcription during photoacclimation , 1997, Photosynthesis Research.

[7]  D. H. Yang,et al.  Regulatory proteolysis of the major light-harvesting chlorophyll a/b protein of photosystem II by a light-induced membrane-associated enzymic system. , 1995, European journal of biochemistry.

[8]  J. Benemann,et al.  Photosystem-II repair and chloroplast recovery from irradiance stress: relationship between chronic photoinhibition, light-harvesting chlorophyll antenna size and photosynthetic productivity in Dunaliella salina (green algae) , 1998, Photosynthesis Research.

[9]  O. Pulz,et al.  Valuable products from biotechnology of microalgae , 2004, Applied Microbiology and Biotechnology.

[10]  P. Joliot,et al.  Quantification of cyclic and linear flows in plants. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Anderson,et al.  The dynamic photosynthetic membrane and regulation of solar energy conversion. , 1988, Trends in biochemical sciences.

[12]  W. Kühlbrandt,et al.  Accumulation of plant antenna complexes is regulated by post-transcriptional mechanisms in tobacco. , 1995, The Plant cell.

[13]  A. McDowall,et al.  Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion. , 2007, Plant biotechnology journal.

[14]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[15]  K Maxwell,et al.  Chlorophyll fluorescence--a practical guide. , 2000, Journal of experimental botany.

[16]  N. Huner,et al.  Redox Regulation of Light-Harvesting Complex II and cab mRNA Abundance in Dunaliella salina , 1995, Plant physiology.

[17]  K. Niyogi,et al.  Non-photochemical quenching. A response to excess light energy. , 2001, Plant physiology.

[18]  I. Ohad,et al.  The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation , 2004, Photosynthesis Research.

[19]  A. Melis,et al.  Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage ? , 1999, Trends in plant science.

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

[21]  Anja Doebbe,et al.  Functional integration of the HUP1 hexose symporter gene into the genome of C. reinhardtii: Impacts on biological H(2) production. , 2007, Journal of biotechnology.

[22]  Olaf Kruse,et al.  Photosynthetic biomass and H2 production by green algae: from bioengineering to bioreactor scale-up. , 2007, Physiologia plantarum.

[23]  T. Masuda,et al.  Truncated chlorophyll antenna size of the photosystems—a practical method to improve microalgal productivity and hydrogen production in mass culture , 2002 .

[24]  S. McKim,et al.  Light‐harvesting complex gene expression is controlled by both transcriptional and post‐transcriptional mechanisms during photoacclimation in Chlamydomonas reinhardtii , 2003 .

[25]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[26]  J. Nickelsen,et al.  NAB1 Is an RNA Binding Protein Involved in the Light-Regulated Differential Expression of the Light-Harvesting Antenna of Chlamydomonas reinhardtii , 2005, The Plant Cell Online.

[27]  K. Niyogi,et al.  A Major Light-Harvesting Polypeptide of Photosystem II Functions in Thermal Dissipation Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002154. , 2002, The Plant Cell Online.

[28]  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.

[29]  T. Ohama,et al.  Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii , 2008, Genetics.

[30]  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.

[31]  Elizabeth H. Harris,et al.  The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use , 1989 .

[32]  E. Stauber,et al.  Proteomics of Chlamydomonas reinhardtii Light-Harvesting Proteins , 2003, Eukaryotic Cell.

[33]  P. Falkowski,et al.  Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Rupprecht,et al.  Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii , 2008, Eukaryotic Cell.

[35]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[36]  Olaf Kruse,et al.  Improved Photobiological H2 Production in Engineered Green Algal Cells* , 2005, Journal of Biological Chemistry.

[37]  Y. Asakura,et al.  Maize Mutants Lacking Chloroplast FtsY Exhibit Pleiotropic Defects in the Biogenesis of Thylakoid Membranes Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.014787. , 2004, The Plant Cell Online.

[38]  P. Horton,et al.  Antisense Inhibition of the Photosynthetic Antenna Proteins CP29 and CP26: Implications for the Mechanism of Protective Energy Dissipation , 2001, Plant Cell.

[39]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J Chory,et al.  The regulation of circadian period by phototransduction pathways in Arabidopsis , 1995, Science.

[41]  Haroon S. Kheshgi,et al.  The Photobiological Production of Hydrogen: Potential Efficiency and Effectiveness as a Renewable Fuel , 2005, Critical reviews in microbiology.