L-homoserine is an important non-proteinogenic amino acid, that has been widely used in the fields of food, feed, pharmaceutical, agricultural and chemical industries. In recent years, microbial fermentation as a promising synthetic route for L-homoserine production has attracted great attention due to its environmentally friendly process. For construction and selection of L-homoserine-hyperproducing strains, a large number of L-homoserine fermentation samples need to be analysed and compared. However, there were few reports on the detection method of L-homoserine up to now. In this study, we coupled cystathionine γ-lyase and lactic dehydrogenase to develop a rapid method for the detection of L-homoserine. After the systematic optimization of the components in the detection system, the best reaction conditions were determined as: cystathionine γ-lyase 12 U/mL, lactic dehydrogenase 50 U/mL, NADH 0.5 mmol/L, PLP 0.1 mmol/L, temperature 40 ℃, pH 7.5. The detection method constructed in this study showed a good liner relationship in the range of L-homoserine of 0.005-0.1 g/L with R2=0.998 2, which provides important technique supports for the analysis of L-homoserine concentration and the breeding of L-homoserine-hyperproducing strain.
ZHAO Jinhua
,
WEI Liang
,
XU Ning
,
MA Zhenping
,
LIU Jun
. Determination of L-homoserine concentration in fermentation broth using double-enzyme coupling method[J]. Food and Fermentation Industries, 2023
, 49(2)
: 239
-245
.
DOI: 10.13995/j.cnki.11-1802/ts.031326
[1] LI H, WANG B S, LI Y R, et al.Metabolic engineering of Escherichia coli W3110 for the production of L-methionine[J].Journal of Industrial Microbiology Biotechnology, 2017, 44(1):75-88.
[2] PARK J H,LEE S Y.Metabolic pathways and fermentative production of L-aspartate family amino acids[J].Biotechnology Journal, 2010, 5(6):560-577.
[3] VAN DAMME M, ZEILMAKER T, ELBERSE J, et al.Downy mildew resistance in Arabidopsis by mutation of homoserine kinase[J].The Plant Cell, 2009, 21(7):2 179-2 189.
[4] XU X S, TENG H B, QIU G F, et al.A facile synthetic route to L-phosphinothricin[J].Chinese Chemical Letters, 2006, 17(2):177-179.
[5] HONG K K, KIM J H, YOON J H, et al.O-succinyl-L-homoserine based C4-chemical production:Succinic acid, homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives, and 1,4-butanediol[J].Journal of Industrial Microbiology & Biotechnology, 2014, 41(10):1 517-1 524.
[6] ZHANG Y J, MA C W, DISCHERT W, et al.Engineering of phosphoserine aminotransferase increases the conversion of l-homoserine to 4-hydroxy-2-ketobutyrate in a glycerol-independent pathway of 1,3-propanediol production from glucose[J].Biotechnology Journal, 2019, 14(9):e1900003.
[7] XU J, SAUNDERS C W, GREEN P W, et al.Microorganisms and methods for producing acrylate and other products from homoserine:US20140099676[P].2014-04-10.
[8] LI Y J, WEI H, WANG T, et al.Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives[J].Bioresour Technology, 2017, 245:1 588-1 602.
[9] XU J Z, HAN M, ZHANG J L, et al.Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway[J].Amino Acids, 2014, 46(9):2 165-2 175.
[10] 王颖珊, 郭峰, 严伟, 等.四碳有机酸生物合成的代谢工程研究进展[J].生物工程学报, 2021, 37(5):1 697-1 720.
WANG Y S, GUO F, YAN W, et al.Advances in the metabolic engineering for the production of tetracarbon organic acids[J].Chinese Journal of Biotechnology, 2021, 37(5):1 697-1 720.
[11] LI H, WANG B S, ZHU L H, et al.Metabolic engineering of Escherichia coli W3110 for L-homoserine production[J].Process Biochemistry, 2016, 51(12):1 973-1 983.
[12] LI N, XU S, DU G C, et al.Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux[J].Biochemical Engineering Journal, 2020, 161:107665.
[13] ZHANG Y, WEI M H, ZHAO G H, et al.High-level production of L-homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering[J].Bioresource Technology, 2021, 327(4):124814.
[14] MU Q X, ZHANG S S, MAO X J, et al.Highly efficient production of L-homoserine in Escherichia coli by engineering a redox balance route[J].Metabolic Engineering, 2021, 67:321-329.
[15] LIU M, LOU J L, GU J L, et al.Increasing L-homoserine production in Escherichia coli by engineering the central metabolic pathways[J].Journal of Biotechnology, 2020, 314-315:1-7.
[16] BRIDGES C C, KRASNIKOV B F, JOSHEE L, et al.New insights into the metabolism of organomercury compounds:Mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase[J].Archives of Biochemistry & Biophysics, 2012, 517(1):20-29.
