An assessment of serial co-cultivation approach for generating novel Zymomonas mobilis strains

Abstract

Objective: The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer, and Z. mobilis-based biorefinery shows great potential for biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if it improves or rewires the desirable physiological features of Z. mobilis, especially ethanol production.

Results: We performed serial co-culture of Z. mobilis with the baker's yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to stress due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains, showing that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. 50 generations of Z. mobilis monoculture did not induce morphological changes or any significant genetic variations. The result indicates that the method needs to be carefully optimized for Z. mobilis strain improvement.

Keywords: Adaptive evolution; Co-culture; Ethanol production; Genomic stability; Zymomonas mobilis.

Fig. 1

Phase contrast images of Z. mobilis monoculture and co-culture with S. cerevisiae strain CEN.PK113-7D or E. coli strain K12. Used strains and conditions are as shown in white texts in the images. Abbreviation glu stands for glucose in the medium. It is to be noted that Z. mobilis strain in the co-culture vs yeast with 100 g/L glucose formed elongated filamentous structure, while other conditions did not induce such drastic morphological changes in Z. mobilis cells. Right-bottom panel and Zm6 vs K12 panel are enlarged images with scale bar 10 μm, while all other panel sizes are corresponding with the scale bar 10 μm, found in the bottom-middle panel. Black a in the right-bottom panel indicates bursting cell with membrane protrusion from the cytoplasm. b The lysed cell exhibiting lighter phase contrast than that of the live cells. c Elongated Z. mobilis cell. d Dead yeast cell

Fig. 2

Characterization of Z. mobilis strains that generated from serial co-culture-based laboratory adaptation. a Total production of acetate, lactate and ethanol from the overnight monoculture of the generated Z. mobilis strains and the parental strain Zm6. b Growth profiles of monoculture Z. mobilis strain obtained in this study and the parental strain Zm6 in the complex medium with glucose 20 g/L. Z. mobilis strain designations are as follows; Zs100; Z. mobilis strain obtained from the last round of Z. mobilis vs S. cerevisiae serial co-culture with glucose 100 g/L, Zs100R; obtained from a parallel replicate of Z. mobilis vs S. cerevisiae glucose 100 g/L, Zs20; Z. mobilis strain obtained from the last round of serial co-culture with S. cerevisiae supplemented with 20 g/L glucose, Zs20R; Z. mobilis strain from a last culture of parallel run of Zs20, Ze20; Z. mobilis strain obtained from last round of Z. mobilis vs E. coli co-culture with 20 g/L glucose, and Ze20R as a parallel replicate of Ze20, Z20 designates Z. mobilis strain obtained from a last round of Z. mobilis monoculture with 20 g/L glucose, and Z100 designates Z. mobilis strain obtained from a last round of monoculture with 100 g/L glucose. Generation time for actively growing Z. mobilis strains in the plate reader was as follows; 175 min for Zm6, 177 min for Zs20, 173 min for Zs100 and 179 min for Ze20. Note that there were no significant differences in ethanol production and growth profiles between strains. Error bars; standard deviations from 3 independent measurements