| Ectoine(1,4,5,6-tetrahydro-2-methyl-4-pyrim-idinecarboxylic acid,ectoine)is a cyclic amino acid derivative.As a compatible solute,it is widely found in halophilic and salt-tolerant bacteria,and can protect and stabilize protein,nucleic acid,biofilm and the whole cell.It has very important commercial value in the fields of cosmetics,enzyme preparation,medical treatment and so on.Therefore,seeking a cheap,efficient and environmental-friendly production method of ectoine in industry has become a research hotspot.In this study,the engineered strain BW/p YB-ect ABC producing ectoine was constructed by overexpressing the ectoine synthetic gene cluster ect ABC and using Escherichia coli BW25113 as the cell substrate.After 9 h reaction using whole-cell conversion technology,the yield of ectoine was 5.94±0.17 m M,the productivity was0.094 g/L/h,and the conversion ratios of aspartic acid and glycerol were 16.25% and11.81%,respectively.Through the optimization of the whole-cell conversion system,the yield of ectoine reached 8.01±0.25 m M,and the productivity was 0.126 g/L/h,which was34.04% higher than that before optimization.The conversion ratios of aspartic acid and glycerol were 18.65% and 13.29%,respectively.However,a byproduct acetic acid reached 14.10 m M.In order to reduce the accumulation of acetic acid and raise the transformation ratio of glycerol,we constructed SG/p YB-ect ABC strain by using SG104(acetic acid metabolic pathway was modified)as the whole-cell chassis.The whole-cell conversion results showed that the yield of ectoine was 9.03±0.09 m M and the productivity was 0.142 g/L/h,which was 12.70% higher than that of BW/p YB-ect ABC strain.And the conversion ratios of aspartic acid and glycerol were 22.23% and 15.35%,respectively.The accumulation of acetic acid was 38.10% lower than that of BW/p YB-ect ABC strain.Therefore,we chose SG104 as the whole-cell chassis for further metabolic modification.In order to strengthen the metabolic flux of the rate-limiting step of aspartic acid to L-ASA in the de-novo synthesis of ectoine,we controlled the metabolic flow of aspartic acid in the conversion process based on SG/p YB-ect ABC.We singly knocked out the 5metabolic branch genes(nad B? ans A? pur A? asp A and pan D)downstream of aspartic acid,and constructed five engineered strains.The whole-cell conversion results showed that the yield of ectoine and the conversion ratios of substrate were lower than that of the original strain.It suggested that the alternative pathway of aspartic acid did not affect the synthesis of ectoine.Then we knocked out the genes of the three metabolic branches downstream of the intermediate product L-ASA(thr A,met L and lys A)to construct three engineered strains.The whole-cell conversion results showed that after 9 h of reaction,the biomass,ectoine production and conversion ratios of SG?lys A/p YB-ect ABC strain were higher than the original strain,the yield of ectoine was 11.47±0.72 m M,and the productivity was 0.181 g/L/h,an increase of 27.46%,the conversion ratios of aspartic acid and glycerol were 26.01% and 16.76%,respectively.Furthermore,the bifunctional aspartic acid kinase that catalyzes aspartic acid to ?-aspartylphosphate were mutated and overexpressed to construct 8 engineered strains using SG?thr A as the chassis based on the spatial structure and domain analysis of Thr A.The results of the whole-cell conversion showed that after 9 h of reaction,the engineered strain SG?thr A/p YB-ect ABC+p RB-Thr A470 and SG?thr A/p YB-ect ABC+p RB-Thr A606 had higher ectoine production and conversion ratios than the original strain.For the strains,the yields of ectoine were 11.17±0.14 m M and 9.96±0.11 m M,and the productivity were0.176 g/L/h and 0.157 g/L/h,which increased by 23.94% and 10.56%,respectively.The conversion ratios of aspartic acid were 34.29% and 27.55%,and the conversion ratios of glycerol were 28.03% and 17.99%.Therefore,we obtained two mutant enzymes Thr A470 and Thr A606 that can normally express aspartyl kinase activity as well as lack homoserine dehydrogenase activity.The conversion ratios and productivity of the strain containing mutant enzyme Thr A470 are higher than those containing the mutant enzyme Thr A606.To further improve the utilization of glucose in the de novo synthesis of ectoine,a high glucose utilization pathway(ET107 strain)was introduced on the basis of the SG104 strain.Meanwhile,the truncated thr A gene was integrated into the genome and the lys A gene was knocked out to construct ET?A?lys A as the new chassis.The engineered strain E.coli ET-ECT01 was constructed by transferring the p YB-ect ABC and p RB-Thr A470 double plasmids.The whole cell conversion results showed that the yield of ectoine was 13.23±0.30 m M,the productivity was 0.209 g/L/h,an increase of 44.41%,and the conversion ratios of aspartic acid and glycerol were 37.88% and 25.49%,respectively.Finally,we applied the high-yielding bacteria E.coli ET-ECT01 for fed-batch fermentation in a 10 L bioreactor.After 48.5 h of fermentation,the yield of ectoine in the fermentation broth reached 41.98 g/L.The productivity is 0.866 g/L/h,the unit cell yield reaches 1.35 g/g DCW,and the glucose molar conversion ratio is 25.76%.Subsequently,we constructed an aspartic acid auxotrophic strain B with ectoine as a nutrient,which can be co-cultured with ectoine high-producing strain.The engineered strain B is supposed to use for the recovery of leaky ectoine in the in-situ purification process. |
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