| L-homoserine（2-amino-4-hydroxybutyric acid）is a non-proteinogenic 4-carbon amino acid that contains a chiral carbon and serves as an important intermediate in biosynthesis.Currently,chemical synthetic pesticides are predominantly used in agricultural production,but they have led to issues such as increasing resistance among pests and severe environmental pollution.Therefore,there is a need to promote and use"green,pollution-free,and healthy"new biological pesticides in agricultural production.L-homoserine is a key intermediate for the synthesis of the biological pesticide L-glufosinate-ammonium.The production methods of L-homoserine mainly include chemical synthesis,enzymatic methods,and microbial fermentation.The limitations of chemical synthesis include expensive reagents,low synthesis efficiency,and tedious purification steps,which limit its production scale and applications.Enzymatic methods require expensive coenzymes and toxic chemical reagents,which limit their industrial production.The advantages of producing L-homoserine through microbial fermentation include low cost,low pollution,and easy control of fermentation conditions.In this study,a method for detecting L-homoserine in fermentation broth was first established,followed by optimization of fermentation conditions,fermentation medium components,and feed strategies using Escherichia coli SIIA-2021 as the starting strain to improve the yield of L-homoserine.The main results of this study are as follows:1.L-homoserine was quantitatively analyzed by HPLC after derivatization with 2,4-dinitrofluorobenzene.An Agilent 1260 HPLC system equipped with a Target-phenyl（II）column was used,with mobile phase A consisting of water:acetonitrile:phosphoric acid =95:5:0.01 and mobile phase B consisting of water:acetonitrile:phosphoric acid=25:75:0.01.Detection was performed at a wavelength of 365 nm,with a column temperature of 35.0℃,a flow rate of 1.2 m L/min,and an injection volume of 5.0μL.The linear relationship between L-homoserine concentration and peak area was good within the range of 50～350μg/m L,with a linear regression equation of Y=7.8662X+31.779（R2=0.9995）.The precision RSD value was 0.12%,the repeatability RSD value was 1.39%,and the average recovery rate of spiked samples was 100.28%.2.A strain of E.coli SIIA-2021 with high L-homoserine production,named E.coli SIIA-2021-13,was separated naturally,and its production yield was found to be 3165.35 μg/m L.Using this strain as a starting strain,the fermentation conditions and composition of the fermentation medium were optimized,and the fermentation conditions were preliminarily determined to be:a fermentation time of 48.0 h,a fermentation temperature of 29.0℃,a shaking speed of 220 rpm,an initial p H value of 7.5,an inoculation rate of 2.0%,and a seed liquid OD600value of 0.6～0.8.The composition of the fermentation medium was preliminarily determined as follows（g/L）:glucose 26.05,yeast extract 1.2,leucine 4.8,Mg SO4·7H2O 1.0,KH2PO41.0,（NH4）2SO414.0,L-lysine 0.2,L-threonine 0.2,Ca CO310.0,and 5.0 m L/L trace element mother liquor.After the optimization of the fermentation conditions for E.coli SIIA-2021-13,the L-homoserine production yield increased by 145.14%to 7759.47μg/m L.3.A pilot-scale study of E.coli SIIA-2021-13 was carried out in a 50 L fermentation tank to determine the best method for adding nutrients to the fermentation medium.The liquid volume in the fermentation tank was 27 L,and the p H was adjusted with concentrated ammonia water to maintain a p H value between 6.9 and 7.1.L-lysine and L-threonine were supplemented to achieve concentrations of 1.0 g/L,1.5 g/L,2.0 g/L when the fermentation time was 12 h,20 h,28 h.During the fermentation process,dissolved oxygen（DO）was controlled at around 30%,and the initial shaking speed was 300 rpm,which was adjusted to a speed-coupled DO later,with an aeration rate of approximately 2.0 vvm.The tank pressure was set to 0.07 MPa 24 hours before and adjusted to 0.09 MPa～0.1 MPa 24 hours later.When glucose was exhausted,sugar was added to the medium,and the glucose concentration was maintained below 5.0 g/L.The highest L-homoserine production yield was achieved when nutrients were added to the fermentation medium at 30 h,34 h,and 38 h,reaching 55932μg/m L. |
