| L-2-aminobutyric acid（L-2-ABA）is an unnatural chiral amino acid,and also the key chiral intermediate material for the synthesis of several important drugs.（S）-2-aminobutanamide prepared by amidation is the key precursor for the preparation of antiepileptic drugs Levetiracetam and Buvaracetam.Apart from this,（S）-2-aminobutanol prepared by reducing L-2-ABA is a key raw material for the anti-tuberculosis drug ethambutol.Leucine dehydrogenase（LDH）is widely used in the preparation of L-2-ABA,but the intermediate product 2-ketobutyric acid（2-OBA）inhibits the catalytic activity of LDH,thus the pathway cannot continue.In this study,leucine dehydrogenase was modified to obtain a mutant to reduce the inhibition of 2-OBA and increase specific enzyme activity.At the same time,the expression of the multi-enzyme cascade system was coordinated through the RBS sequence to solve the problem of rate imbalance for more efficient synthesis of L-2-ABA.The main contents are summarized as follows:（1）The leucine dehydrogenase gene derived from Exiguobacterium sibiricum was cloned on the expression vector p ET28a,then successfully expressed in E.coli.The specific enzyme activity was 266 U·mg-1,the optimum temperature and pH were 55℃and 9.0;（2）In view of the obvious inhibition of LDH by 2-OBA and low enzyme activity,a reasonable high-throughput screening method was established to screen mutants with high tolerance to 2-ketobutyric acid and high enzyme activity.A mutant X with specific enzyme activity increased by 31%was obtained;Construct site-directed mutants to verify the beneficial mutation of mutant X,and mutant K72A was chosen whose specific enzyme activity was increased to 133.3%and the half-inhibitory concentration was increased to 14 g·L-1.A site-saturation mutant library at K72 site was constructed,and the enzyme activity of K72A and K72W were found significantly high,and the pH stability of the mutant K72A was more stable and kcat·Km-1 was 2.65 times of the wild type;（3）Design RBS sequences of different intensitys to weaken the expression of threonine deaminase and construct a multi-enzyme cascade recombinant strain of threonine deaminase,LDH and formate dehydrogenase.The recombinant strain E.coli BL21/p ET28a-R3ivl A-EsldhK72A-fdh was obtained,and its yield of L-2-ABA was 2.6 times that of the original strain and the molar conversion rate was 88.8%.The whole-cell biotransformation conditions of the recombinant strain E.coli BL21/p ET28a-R3ivl A-EsldhK72A-fdh were optimized.It showed that the optimal temperature and pH were 35℃and 7.5,respectively,and the optimal substrate concentration was 150 g·L-1.At this time,the output of L-2-ABA was 115.8 g·L-1,and the molar conversion rate was 90.8%.Moreover,E.coli BL21/p ET28a-R3ivl A-EsldhK72A-fdh was cultured for whole-cell biotransformation in 5 L fermentor under optimal conditions.1 L conversion system was fed with about 150 g L-threonine,and the yield of L-2-ABA was the highest at 24 h with 120.8 g L-2-ABA,5.2 g 2-OBA.At this time,the molar conversion rate was 94.8%.This is the highest yield reported for L-2-ABA produced by single cell. |
