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Bioprocess and biosystems engineering

Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance.


PMID 25492311

Abstract

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30xa0% of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5xa0gxa0L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5xa0gxa0L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117xa0gxa0L(-1) at txa0=xa066xa0h and 32530xa0Uxa0L(-1) at txa0=xa053xa0h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at txa0=xa053xa0h as 0.21xa0gxa0g(-1) in S5 strategy.