Producing Strain Construction And Fermentation Condition Optimization Of 2-Aminobutyrate

L-aminobutyrate, a nonnatural amino acid, is widely used in the chemical and pharmaceutical industries, such as levetiracetam and ethambutol. In this article, in order to construct L-2-aminobutyrate producing strains, L-threonine overproducing strain Escherichia coli THRD was used as original strain under the guidance of metabolic engineering theory.To construct 2-ketobutyrate producing strains named as THRD(pTrc99a-TPilv A4) and THRD(pTrc99a-ilvA), the threoine dehydratase encoding genes ilvA4 and ilvA whose feedback inhibition were removed were overexpressed into Escherichia. coli THRD. The result of flask fed-batch fermentation revealed that, THRD(pTrc99a-TPilvA4) and THRD(pTrc99a-ilvA) could convert L-threonine to 2-ketobutyrate, with a production of 8.3±0.4 g/L and 9.23±0.31 g/L, and a yield on glucose of 13.83% and 15.38% respectively, while the control strain could scarcely accumulate 2-ketobutyrate. Further, THRD(pTrc99a-ilvA) could produced 18 g/L of 2-ketobutyrate in the 5 L fed-batch fermentation THRB, THRG1, THRG2 and THRL, the L-2-aminobutyrate producers, were constructed by coexpressed ilvA Wtih tyrB, GDH and bcdBS respectively. The result of flask fed-batch fermentation revealed that, THRB, THRG2, THRG1 and THRL were able to produce 4.07±0.16 g/L,5.81±0.56 g/L,8.99±1.1 g/L and 7.87±0.44 ·g/L of L-2-aminobutyrate with a yield on glucose of 6.78%,9.68%,14.98% and 13.11%, respectively. The accumulation of 19 g/L L-2-aminobutyrate by THRL showed a greater than other strains in L-2-aminobutyrate fermentation. These results demonstrated that rational modification of threonine pathway in E. coli THRD could efficiently overproduce nonnatural L-2-aminobutyrate.Due to the fact that over-accumulation of L-threonine by E.coli THRD has a great relationship with threonine transport, the modification of L-threonine transportation system can reduce the over-secretion of L-threonine. Hence, the single gene deletion mutant THRLArhtA and THRL△rhtC, and double genes deletion mutant THRL△rhtAArhtC were constructed to reduce the over-secretion of L-threonine. The results of flask fermentation showed that concentration of L-threonine by THRLArhtA and THRL△rht C decreased by 47.03% and 44.86% respectively and L-2-aminobutyrate increased by 71.28% and 42.44% (13.48±0.84 g/L.11.21±0.38 g/L VS.7.87±0.44 g/L) respectively compared with control strain. Moreover, THRL△rhtA and THRLArhtC achieved a 10.42% and 4.7% higher yield of L-2-aminobutyrate in 5 L fed-batch fermentation. While, the double mutant THRL△rhtAArhtC produced less L-threonine than THRL, accompanying by a less accumulation of L-2-aminobutyrate. The experimental results showed that the appropriate modification of L-threonine transport system was conductive to L-2-aminobutyrate accumulation. However, double gene deletion resulted in decrease of L-2-aminobutyrate accumulation.THRLC1 and THRLC2, owning two L-2-aminobutyrate pathways, were constructed by introducing citramalate pathway of M. jannaschii into THRL. Compared with the control strain, THRLC1 and THRLC2 accumulated similar production of 2-ketobutyrate and increased 29.23% and 34.77% of L-2-aminobutyrate production respectively. The experimental results showed that the synergy between pathways could efficiently increased L-2-aminobutyrate. Utilization of such pathways in combination leads to an increased metabolite productivity and yield compared to using L-threonine pathway alone.