Whole-cell Biotransformation System For Synthesis Of L-2-aminobutyric Acid And D-gluconic Acid In Recombinant Escherichia Coli

L-2-aminobutyric acid and D-gluconic acid have been widely used in food and pharmaceutical industry.A whole-cell catalyst using Escherichia coli BL21?DE3?as a host,expressing L-threonine dehydratase?LTD?from Escherichia coli K-12,and co-expressing leucine dehydrogenase?LDH?from Bacillus cereus ATCC 14579 and glucose dehydrogenase?GDH?from Bacillus subtilis 168 for cofactor regeneration,has been successfully constructed and used for one-pot production of L-ABA and D-gluconic acid with L-threonine and D-glucose.Have got two recombinant strains BL21/p ET28a-ltd and BL21/p ET28a-ldh/gdh.The LTD,LDH and GDH were successful expressed in E.coli BL21 by IPTG induced.The crude enzyme of LTD,LDH and GDH enzyme activity was 23.28,3.88 and 9.73 U/m L.Compared with the cell-free system,whole-cell catalyst is more stable,has more advantage on repeat utilization,and has higher production efficiency,is a cost-attractive alternative.Enzymatic properties of LTD and LDH was studied.Reaserch of enzymatic properties showed that the LTD optimal temperature and p H were 50?and 9.5.LTD is stable under temperature 30?and between p H 7.0⁹.0.Metal ions and EDTA had no effect on LTD enzyme activity.LDH optimal temperature and p H were 55?and 9.5.LTD is stable under temperature 40?and between p H 7.0⁹.0.EDTA had no effect on LTD enzyme activity and Mn²⁺,Ca²⁺,Mg²⁺can increase enzyme activity in small range.The catalytic condition of whole-cell system was studied in shake flask,including temperature,p H,proper permeabilization of cells and optimal cells amount.The results showed that optimal temperature and p H were 30?and 7.5,triton x-100 is an applicable permeabilization medium,and optimal concentration was 0.3%.The optimum proportion of BL21/p ET28a-ltd and BL21/p ET28a-ldh/gdh was 1:5,and optimum cells amount was 10 g/L.Then the whole-cell catalyst production of L-ABA and D-gluconic acid was carried out in shake flask,148 g/L L-threonine and 224 g/L D-glucose were converted to 125.5 g/L L-ABA and 238.4 g/L D-gluconic acid,the substrate molar yield is 98%.Furthermore,the scale up of was carried out in a 5 L fermentor,the effect of dissolved oxygen on cell growth was studied.The results showed that suitable dissolved oxygen has important influence on cell growth.Then the whole-cell catalyst production of L-ABA and D-gluconic acid was carried out in 5 L fermentor,164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid with productivity of 7.1g/?L?h?and 13.5 g/?L?h?,the substrate molar yield is 99%.The cell repeat utilization was studied,the results showed the recombinant cell can only single repeat in high efficiency.