L-2-aminobutyrate is a critical chiral precursor of new drugs, which has been widely used in chemical and pharmaceutical industries. In this study, the metabolic pathway of L-threonine in Escherichia coli THRD was extended to obtain an L-2-aminobutyrate producing strain. The threonine dehydratase encoding genes ilvA2 and ilvA4 were firstly individually overexpressed in E. coli THRD. The resulting strain THRD/pTrc99a-ilvA2 produced 18 g/L L-2-ketobutyrate in a 5 L fermenter by fed-batch fermentation. Subsequently, encoding genes of tyrosine aminotransferase, glutamate dehydrogenase, and leucine dehydrogenase, tyrB, gdh, and bcdBS, respectively, were overexpressed together with ilvA2 to catalyze the conversion of L-2-ketobutyrate to L-2-aminobutyrate. The strain THRD/pTrc99a-bcdBS-ilvA2 produced 19 g/L L-2-aminobutyrate. The effects of disrupting L-threonine exporters on L-2-aminobutyrate fermentation were investigated, and the production of L-2-aminobutyrate in strain THRDΔrhtC/pTrc99a-bcdBS-ilvA2 increased to 22 g/L. Taken together, the results clearly indicated that the L-threonine producing strain could be effectively transformed into an L-2-aminobutyrate producer by extending its downstream metabolic pathway. This study lays a solid basis for constructing L-2-aminobutyrate high producing strains. This study can also be referred in other metabolic engineering studies to biosynthesize new products by pathway extension.
WANG Ting
,
HAN Chao
,
MAO Qian
,
ZHANG Dezhi
,
CAI Ningyun
,
LIU Hongliang
,
LI Yanjun
,
CHEN Ning
. Construction of Escherichia coli strain producing L-2-aminobutyrate[J]. Food and Fermentation Industries, 2019
, 45(3)
: 56
-63
.
DOI: 10.13995/j.cnki.11-1802/ts.018611
[1] 焦庆才,刘均忠,陈争依,等.L-2-氨基丁酸酶法转化制备:中国,101538596[P].2009-09-23.
[2] AKHTERUZZAMAN S,KATO Y,KOUZUKI H,et al.Carrier-mediated hepatic uptake of peptidic endothelin antagonists in rats[J]. Journal of Pharmacology and Experimental Therapeutics,1999,290(3):1 107-1 115.
[3] HALE K J,CAI J,MANAVIAZAR S,et al.Synthetic studies on the azinothricin family of antibiotics. Part 4. Enantioselective synthesis of the northern half of antitumour antibiotics A83586C and citropeptin[J].Tetrahedron Letters,1995,36(38):6 965-6 968.
[4] 郭勇,王力.一种新的制备单一构型的2-氨基丁酸或其衍生物的方法:中国,CN1510025[P].2004-07-07.
[5] 李国龙.酶法转化制备L-2-氨基丁酸[D].武汉:湖北大学,2017.
[6] CHOI H S,LEE S Y,KIM T Y,et al.In silico identification of gene amplification targets for improvement of lycopene production[J].Applied and Environmental Microbiology,2010,76(10):3 097-3 105.
[7] SHEN C R,LIAO J C.Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways[J].Metabolic Engineering,2008,10(6):312-320.
[8] CHEN L,CHEN Z,ZHENG P,et al.Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli[J].Applied Microbiology and Biotechnology,2013,97(7):2 939-2 949.
[9] PARK J H,OH J E,LEE K H,et al.Rational design of Escherichia coli for L-isoleucine production[J].ACS Synthetic Biology,2012,1(11):532-540.
[10] ZHANG C L,QI J S,LI Y J,et al.Production of α-ketobutyrate using engineered Escherichia coli via temperature shift[J].Biotechnology and Bioengineering,2016,113(9):2 054-2 059.
[11] DANCHIN A,DONDON L,DANIEL J.Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12[J].Molecular and General Genetics,1984,193(3):473-478.
[12] LEE K H,JIN H P,KIM T Y,et al.Systems metabolic engineering of Escherichia coli for L-threonine production[J].Molecular Systems Biology,2007,3(1):http://msb.embopress.org/content/3/1/149.
[13] DIESVELD R,TIETZE N,FÜRST O,et al.Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production[J].Journal of Molecular Microbiology and Biotechnology,2009,16(3/4):198-207.
[14] 张雪,温廷益.Red重组系统用于大肠杆菌基因修饰研究进展[J].中国生物工程杂志,2008,28(12):89-93.