Development of Stable Flocculent Saccharomyces cerevisiae Strain for Continuous Aspergillus niger -Galactosidase Produc

1. Development of Stable Flocculent Saccharomyces cerevisiae Strain for Continuous Aspergillus niger �-Galactosidase Production Carla Oliveira, Jos� A. Teixeira, Nelson Lima, Nancy A. Da Silva, and Luc�lia Domingues

2. INTRODUCTION �-Galactosidase �-Galactosidase can cleave �-linked galactose residues from various compounds Commonly used to cleave lactose Widely used in the food and pharmaceutical industries

3. INTRO�� Plasmid loss in a nonselective medium led to a decrease in productivity during continuous culture operation A continuous high cell-density system leading to increased volumetric productivity The utilization of flocculating cells provides an important contribution to the improvement of downstream processing Strain stability is needed so that continuous operation does not lead to a progressive loss of the product Integrating a recombinant DNA into the genome

4. INTRO�� To increase the number of the integrated copies of the desired gene use repetitive sequences ? Target sites for homologous recombination The d-sequences flank the regions of the Ty1 retrotransposon of S. cerevisiae Allowing multiple gene copy integration BIG PROBLEM ? Looped out through excisional recombination

5. MATERIALS AND METHODS Strains and plasmids: The lacA expression cassette containing the lacA gene flanked by the ADH1 promoter and terminator was isolated from plasmid pVK1.1

6. MATERIALS AND METHODS Plasmid pd-neo was used as carrier vector for the integration of the lacA expression cassette into the yeast chromosomes d-integrating vector containing the bacterial neo resistant gene for selection against G418

7. MATERIALS AND METHODS Plasmid construction:

8. MATERIALS AND METHODS Yeast transformation: By electroporation Selection against G418 antibiotic To select for �-galactosidase producing colonies, YPD medium was supplemented with 40 �g/ml (X-gal).

9. MATERIALS AND METHODS �-galactosidase activity: Measuring the release of p-nitrophenol from p-nitrophenyl-�-D-galactopyranoside ( pNPG) Absorbance was measured at 405 nm

10. MATERIALS AND METHODS Southern blot analysis: Integration copy number and patterns of the �-galactosidase expression cassette were analysed Total genomic DNA ? digested with XbaI ? agarose gel electrophoresis ?nylon membrane ? Cross Linking ? Probes were labelled ? measuring the relative ratio of intensities of the multicopy band to a single-copy band

11. RESULTS Chromosomal integration of lacA gene : A linearized pCO1 plasmid was introduced into S. cerevisiae The number of transformants obtained was largely dependent on the G418 concentration added Longer incubation after yeast transformation prior to G418 selection was extremely important for transformant recovery The colonies with a deep blue colour, expressing higher �-galactosidase activity, were screened by a standard �-galactosidase activity assay

12. RESULTS Chromosomal integration of lacA gene :

13. RESULTS Characterization of integrated gene expression: Linear relationship can be observed up to a copy number of eight

14. RESULTS Stability evaluation of recombinant strains : Eight sequential batch cultures ? Spread on YPD plates ? Analysis of G418 and X-gal ? Southern blot analysis Copy number maintened !!?

15. RESULTS Continuous-culture experiments: During the continuous cultures,the percentage of cells expressing �-galactosidase was higher for the integrant strain than for the strain carrying the plasmid pVK1.1

16. RESULTS Continuous-culture experiments: For the plasmid-carrying strain, 60% white colonies were observed in the X-gal plates For the integrant strain, all colonies were counted as �-galactosidase producing cells

17. DISCUSSION The use of the d-integration system enable increased production of heterologous proteins A linear correlation between �-galactosidase activity and integrated copy number It indicates that the integration on different d-sites does not alter gene expression For the integration of multiple gene copies, it is extremely important to check for stability as the integrated copy number may decrease by gene loop-out events

18. Conclusion construction of S. cerevisiae strains producing extracellular A. niger �-galactosidase at levels comparable to the multicopy Yep plasmid system. For the continuous high cell-density culture, the integrant strain was more stable and presented a higher specific �-galactosidase activity than the plasmid-carrying strain