The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.
BACKGROUND
Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein.


METHODS
Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells.


RESULTS
The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced cultures) or already reverted to a non-producing cells (in long-term photobioreactor cultivations), emphasizing the sensitivity and resolution of the method.


CONCLUSIONS
The fluorescence microscopy method displays a useful technique for a rapid detection of non-producing single cells in an ethanol producing cell population.
Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Bahnhofstraße 1, 16-2001, D-15745, Wildau, Germany. kschulze@th-wildau.de.
Katja
K
Schulze
Algenol Biofuels Germany GmbH, Berlin, Germany. Imke.Lang@algenol.com.
Imke
I
Lang
Algenol Biofuels Germany GmbH, Berlin, Germany. Heike.Enke@algenol.com.
Heike
H
Enke
Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Bahnhofstraße 1, 16-2001, D-15745, Wildau, Germany. diana.lopez@th-wildau.de.
Diana
D
Grohme
Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Bahnhofstraße 1, 16-2001, D-15745, Wildau, Germany. mfrohme@th-wildau.de.
Marcus
M
Frohme
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D056804:Biofuels; D015415:Biomarkers / Q000032:analysis; D003116:Color; D000431:Ethanol / Q000378:metabolism; D005453:Fluorescence; D042822:Genomic Instability; D007091:Image Processing, Computer-Assisted / Q000379:methods* / Q000706:statistics & numerical data; D060847:Metabolic Engineering; D008856:Microscopy, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D057054:Molecular Imaging / Q000379:methods* / Q000706:statistics & numerical data*; D010798:Phycocyanin / Q000032:analysis; D011767:Pyruvate Decarboxylase / Q000235:genetics / Q000378:metabolism; D046940:Synechococcus / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D019076:Transgenes
D056804:Biofuels; D015415:Biomarkers; D010798:Phycocyanin; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D011767:Pyruvate Decarboxylase