一株高产丝氨酸羟甲基转移酶基因工程菌的构建及发酵产酶条件优化

Construction of and optimization of fermentation conditions for a genetically engineered strain that yields high serine hydroxymethyltransferase

以E.coli AB90054基因组中DNA为模板,采用PCR方法扩增得到glyA基因,将该基因克隆到表达载体pET28a(+)中,转化表达菌株BL21(DE3)后筛选阳性重组子,并对筛选所得高表达基因工程菌G16进行培养条件和表达条件的优化。结果表明,当培养条件为38℃、pH为7.0、转速为150r/min时,基因工程菌的生长速度和菌液浓度最佳;当表达条件的诱导温度为30℃、诱导剂浓度为0.5mmol/L、诱导时间为5h时,基因工程菌诱导目的蛋白表达效果最好。通过SDS-PAGE分析和酶活测定后发现,在优化条件下基因工程菌G16发酵产丝氨酸羟甲基转移酶能力占菌体总蛋白的60%左右,且蛋白活性正常。

A gene （glyA） encoding serine hydroxymethyltransferase （SHMT） from Escherichia coli strain AB90054 was amplified and sequenced, which consists of 1254 base pairs and whose sequence is exactly the same with the glyA gene in E. coli K-12 that had been reported. This gene was inserted into the expression vector pET28a （＋） and pET28a-glyA was obtained. Then the recombinant plas- mid was introduced into expression strain BL21 （DE3）, and positive recombinants were selected for induced expression after PCR confirmation. Optimization of culture conditions and expression condi- tions were performed on genetically engineered strain G16 which possesses the highest SHMT expres- sion in screening assay. Results of fermentation conditions show that best growth of the engineered bacteria and bacterial concentration can be achieved under the culture conditions of incubation temper- ature 38 ℃, initial pH of medium 7.0 and rotation speed of table concentrator 150 r/min; best target protein expression can be induced under the expression conditions of inducing temperature 30℃ and when the bacteria are induced for 5 hours by 0.5 mmol/L IPTG. SDS-PAGE analysis and enzyme as- say show that the genetically engineered bacteria G16 can efficiently express serine hydroxymethyl- transferase protein with SHMT catalytic activity and reach about 60% of the total bacterial protein under optimized fermentation conditions.