Codon optimization of the calf prochymosin gene and its expression in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> |
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Authors: | Zhen Feng Lanwei Zhang Xue Han Yanhe Zhang |
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Institution: | (1) College of Food Science, Northeast Agricultural University, 59 Mucai Road, 150030 Harbin, Heilongjiang, People’s Republic of China;(2) College of Food Science and Technology, Harbin Institute of Technology, 202 Haihe Road, 150090 Harbin, Heilongjiang, People’s Republic of China; |
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Abstract: | Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were
obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient
translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping
the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask
culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared
with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass
of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf
chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production. |
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