Construction Of L-threonine-producing Strains And Optimization Of Fermentation Process

L-threonine,an essential amino acid,is widely used in the food,pharmaceutical and feed industries.In Escherichia coli fermentation process,oxaloacetic acid as a precursor participates in the synthesis of L-threonine,so by increasing the amount of precursor oxaloacetic acid,the production of L-threonine can be improved.In addition,increasing the intracellular concentration of NADPH,can provide sufficient reducing equivalents in L-threonine synthesis approach,which will further improve high yield for L-threonine production.Adding betaine in the medium and optimizing culture conditions have a positive effect on enhancing the conversion rate of L-threonine.In L-threonine fermentation process,the glyoxylate shunt is thought to function as part of the anaplerotic pathway.In this study,we constructed E.coli THRD ?iclR,THRD PI and THRD P2 via knockout and replacement of aceBAK promoter in E.coli THRD,an L-threonine producer.Batch culture of engineering strain E.coli THRD ?iclR resulted in a production of 42.60 ±1.23 g/L L-threonine and a yield of 32.77%glucose,which was respectively 20.61%and 20.7%higher than that of the control strain THRD.The titer of L-threonine in E.coli THRD P1 culture reached 36.50±1.42 g/L and the yield reached 28.08%glucose,which was 3.34%and 3.39%higher than that obtained from the control strain,respectively.However,the inhibition of cell growth occurred after 8 h in E.coli THRD P2 and resulted in a production of 8.31±1.31 g/L L-threonine and a yield of 20.78%glucose,which was respectively 76.47%and 23.52%lower than that of the control strain THRD.In conclusion,moderate modification of the glyoxylate shunt benefited the L-threonine production;however,excessive enhancement of glyoxylate shunt had a negative effect on the normal cell metabolism.Pyruvate carboxylase catalysis reaction is the major approach to supply the oxaloacetate,but it does not contain this enzyme in Escherichia coli.Pyruvate carboxylase from Bacillus subtilis was expressed heterologously in E.coli THRD,an L-threonine producer.Batch culture of engineered strain E.coli THRD +pWSK29-pycA resulted in a production of 46.09 ±1.58 g/L L-threonine and a yield of 30.72%glucose,which was respectively 12.28%and 12.24%higher than that of the control strain E.coli THRD+pWSK29.The titer of L-threonine in E.coli THRD+pTrc99a-pycA culture reached 35.91 ±1.15 g/L and the yield reached 23.94%glucose,which was 10.36%and 10.37%lower than that obtained from the control strain,respectively.However,compared to the control strain,E.coli THRD+pTrc99a-pyc A increased 31.690/%on biomass.The results showed that appropriate expression of pyruvate carboxylase was conducive to the synthesis of L-threonine.After the end of fermentation,The constructed E.coli THRD pykF::pycA of L-threonine production did not change significantly compared to E.coli THRD ?pykF,indicating based on knockout the gene pykF,heterologous expression pycA gene had little effect on the accumulation of L-threonine.NADPH is an important intracellular reducing equivalents.Increasing the supply of NADPH in the L-threonine engineering bacteria which depend NADPH as a coenzyme can promote the synthesis product and optimize the performance of strains.In this experiment,We constructed a recombinant E.coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum,encoded by the gapC.Glyceraldehyde-3-phosphate dehydrogenase plays an important role in the glycolytic pathway in E.coli.The experiments showed that the number of intracellular NADPH synthesis were improved.The results of flask fed-batch fermentation revealed that,THRD gapA::gapC produced slightly higher L-threonine than the original strain THRD.Further,THRD gap A::gapC could produced 119.53 g/L of L-threonine and a yeid on glucose of 49.51%in the 5 L fed-batch fermentation,increased by 8.68%and 8.67%compared with control strain(109.98 g/L,45.56%).In summary,substitution the key enzyme of the glycolytic pathway,increased intracellular NADPH,was conducive to L-threonine synthesis.Betaine as an alkaloid that can regulate intracellular osmotic pressure and provide methyl in the synthesis.In this experiment,betaine hydrochloride and phosphate were added in the culture medium.Fermentation results indicated that it was most obvious that initially adding 0.5 g/L betaine hydrochloride and subsequently feeding it at a rate of 1 g/L with glucose and the final conversion rate could reach 54.94%increasing by 15.06%compared with the control strain THRD(47.75%).