基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

Impact of gene dosage on recombinant transglutaminase production of Pichia pastoris

摘要:

基于毕赤酵母核糖体DNA序列 (rDNA)，构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg，并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115，得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数，进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明，被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c)，其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c；高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c，发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重，其中单位菌体酶活mtg-3c是mtg-6c的1.4倍；mtg-3c纯化酶的最高酶活达到7.21 U/mL，蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响，发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达，MTG高酶活与菌株较高分泌蛋白有关。

Abstract:

Based on the rDNA sequence of Pichia pastoris, a multi-copy gene expression vector of transglutaminase (pPICZα-rDNA-mtg) was constructed and transformed to the host strain (pGAP9-pro/GS115) expressing pro peptide, to obtain the co-expression strain pro/rDNA-mtg (GS115). Real-time fluorescence quantitative PCR (qPCR) was used to analyze transglutaminase gene copy number in the 4 positive strains. We further studied the effect of gene copy on the enzyme production of recombinant Pichia pastoris as well as high-density fermentation of higher expression strain in a 3-L fermenter. The mtg copy numbers of the 4 positive strains were 2.21, 3.36, 5.72 and 7.62 (mtg-2c, mtg-3c, mtg-6c and mtg-8c), respectively, and the enzyme production capacity and protein expression level were mtg-3c>mtg-2c>mtg-6c>mtg-8c. Mtg-3c and mtg-6c of high-density fermentation had the highest enzymatic activity and enzymatic activity per unit wet weight in the supernatant of 3.12 U/mL, 52.1 U/g (wet weight) and 2.07 U/mL and 36.5 U/g (wet weight), respectively. In terms of enzyme activity per unit wet weight, mtg-3c is 1.4 times higher than that of mtg-6c. The activity of purified enzyme (mtg-3c) was up to 7.21 U/mL and the protein concentration was 437.2 μg/mL. By analyzing the effect of mtg copy number on the enzyme production of recombinant strains, mtg-3c is suitable for the co-expression of two genes (pro and mtg) in pro/rDNA-mtg, and its enzyme activity is related to higher protein secretion of the strain.