Construction Of Acid And Heat-resistant Threonine Strain

L-threonine,one of all the essential amino acids,as an important component of protein,is widely used in food,feed and pharmaceutical industries.In the process of Escherichia coli fermentation,maintaining low pH and high fermentation temperature can save the dosage of liquid ammonia and cooling water.If the fermentation is finished at low pH,for extraction process such as concentration and electric crystallization,sulphuric acid can be saved.Therefore in the process of fermentation controlling pH and temperature is helpful to reduce the energy consumption of amino acids production.In this paper,when the acid-resisting system(ARs)of E.coli is modified,E.coli can survive in acidic environment.When small heat shock protein HSP20 and ectoine synthesis pathway are introduced,E.coli can improve the heat-resisting property.So L-threonine producing strain E.coli THRD can obtain higher yield of L-threonine under the condition of low pH and high temperature.With L-threonine-producing strain THRD as starting strain,by modifying its AR2 or AR3 acid-resisting system,the strains THRD(pTrc99a-gadBC),THRD araABD::gadBC,THRD(pTrc99a-LGOX),THRD(pTrc99a-arcA)and THRD araABD::arcA were constructed.The culture manner of 5 L fed-batch fermentation of acid-resisting system modified strains was that the pH before 24 h of the fermentation was maintained at 7.0.After 24 h of the fermentation,ammonium solution was not fed,and pH was not adjusted before pH reached 6.0.The pH was maintained at 6.0 until the end of fermentation.At the end of the fermentation,L-threonine production of THRD(pTrc99a)reached 100±1g/L and sugar acid conversion rate reached 32.9%.L-threonine production of THRD(pTrc99a-gadBC),THRD(pTrc99a-arcA)and THRD(pTrc99a-LGOX)reached 106±2 g/L,115±3 g/L and 115±2 g/L respectively,the sugar acid conversion rate reached 34.7%,36.9%and 36.5%respectively,and compared with THRD(pTrc99a)L-threonine production and conversion rate of the modified stains increased by 6.0%,6.0%,10.0%and 5.6%,10.0%,10.9%respectively.E.coli THRD araABD::gadBC and THRD araABD::arcA grew slowly after 12 h of the fermentation so the biomass and L-threonine production decreased.The fermentation results showed that L-threonine production of several acid-resisting strains whose AR2 and AR3 acid-resisting systems were modified was improved in acidic conditions.With L-threonine-producing strain THRD as starting strain,by modifying its heat-resisting system,the strains THRD(pTrc99a-ectABC)and THRD(pBV220-hsp20)were constructed.The culture manner of 5 L fed-batch fermentation of the heat-resisting system modified strains was that initial fermentation temperature was 37? in earlier middle fermentation temperature was controlled at 37? and about 12-14 h namely OD600(30-35),the temperature was adjusted to 40? and stayed unchanged continuing fermentation until the end.At the end of the fermentation,L-threonine production of THRD(pBV220-hsp20)reached 129 g/L,sugar acid conversion rate reached 37.1%,and compared with THRD(pBV220)L-threonine production and acid sugar conversion rate increased by 7.5%and 6.9%respectively.Biomass of THRD(pTrc99a-ectABC)was 15.58 g DCW/L and THRD(pTrc99a)was 11.97 g DCW/L.L-threonine production of THRD(pTrc99a-ectABC)reached 125 g/L,sugar acid conversion rate reached 40.3%,and compared with THRD(pTrc99a)L-threonine production and acid sugar conversion rate increased by 9.60%and 11.14%respectively.The results showed that overexpression of genes ectA?ectB?ectC and hsp20 are beneficial to increase L-threonine production under the condition of high temperature fermentation.