For production of HA with high molecular weight, yeast has been engineered. Saccharomyces cerevisiae and Pichia pastoris have been used as a host due to advantages of large UDP-sugars’ pool and easy genetic manipulation. The codon-optimized genes of hyaluronan synthase and UDP-glucose dehydrogenase form Xenopus laevis were expressed in S. cerevisiae and P. pastoris. The transcription of genes has been confirmed by RT-PCR. The concentration of hyaluronic acid from recombinant P. pastoris was about 200mg/L while that from recombinant S. cerevisiae was about 150mg/L. The recombinant P. pastoris was thus chosen to as a suitable host for HA production.
The genes of HA pathway have been overexpressed by several combinatorial expression cassettes. EJP strain overexpressing HA pathway genes has been established. EJP-D, EJP-CD and EJP-CDE cells have been further constructed, by introducing more copied of the HA expression cassette and produced 1.6g/L HA with 1.2MDa, 1.7g/L with 1.2MDa, and 1.2g/L with 1.3MDa, respectively.
The hyaluronan synthase from X. laevis can produce HA in MW raging to spanning to 10MDa in in vitro in presence of sufficient UDP-sugars. To produce HA with higher molecular weight, the expression level of hyaluronan synthase has been altered by replacement of AOX1 promoter with AOX2 promoter. The AOX2 promoter has one-tenth the activity of the AOX1 promoter. The EJW, EJWP-CD, and EJWP-CDE cells produced HA with 1.5MDa, 2.1MDa and 2.0MDa, respectively. The MW of HA from EJW series cells is much higher than one (1.2MDa) obtained from EJP cells. On the other hand, total production amounts of HA from EJW series cells were smaller than these obtained with EJP cells. Due to the reduced expression of hyaluronan synthase on the cell membrane, the total HA yield was decreased.
Low-temperature cultivation has a meaningful effect on the MW of HA. The MW of HA is produced at various temperatures using EJWP-CDE cells. In the experiments, cultivation of the cells started at 30℃ following a glucose feeding phase at the same temperature. Then, the temperature was changed at the induction phase. As expected, the size of the HA polymer was dependent on temperature. EJWP-CDE cells which were cultured at 26℃ produced the highest MW of over 2.5MDa, while the cells that were cultured at 30℃ produced the lowest MW of 2MDa. The result indicates that low-temperature cultivation helps to flux from cell wall synthesis to HA synthesis.