L-2-氨基丁酸大肠杆菌生产菌株的构建
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摘要
L-2-氨基丁酸作为新型药物的关键手性前体,在化工和制药行业应用广泛。该文以1株生产L-苏氨酸的大肠杆菌(Escherichia coli) THRD为出发菌株,逐步延伸代谢途径,构建了L-2-氨基丁酸高产菌株。首先,分别把苏氨酸脱水酶编码基因ilv A2和ilv A4在THRD中过表达,菌株THRD/p Trc99a-ilv A2在5 L发酵罐中分批补料发酵,2-酮基丁酸积累量达到18 g/L。然后,分别与ilv A2串联表达酪氨酸转氨酶、谷氨酸脱氢酶和亮氨酸脱氢酶编码基因tyr B、gdh和bcdBS,将L-2-酮基丁酸转化为L-2-氨基丁酸,菌株THRD/p Trc99a-bcdBS-ilv A2的L-2-氨基丁酸产量达到19 g/L。最后,研究了阻断L-苏氨酸输出途径对发酵的影响,菌株THRDΔrht C/p Trc99a-bcdBS-ilv A2的L-2-氨基丁酸产量提升至22 g/L。因此,通过代谢途径延伸可以有效地将L-苏氨酸生产菌株转变为L-2-氨基丁酸生产菌株。该研究为L-2-氨基丁酸高产菌株的构建奠定了基础,且对其他延伸代谢途径获得新产品的代谢工程研究提供了参考。
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 ilv A2 and ilv A4 were firstly individually overexpressed in E. coli THRD. The resulting strain THRD/pTrc99 a-ilv A2 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 ilv A2 to catalyze the conversion of L-2-ketobutyrate to L-2-aminobutyrate. The strain THRD/p Trc99 a-bcdBS-ilv A2 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/pTrc99 abcdBS-ilv A2 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.
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 ilv A2 and ilv A4 were firstly individually overexpressed in E. coli THRD. The resulting strain THRD/pTrc99 a-ilv A2 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 ilv A2 to catalyze the conversion of L-2-ketobutyrate to L-2-aminobutyrate. The strain THRD/p Trc99 a-bcdBS-ilv A2 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/pTrc99 abcdBS-ilv A2 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.
引文
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[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 ofα-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,FRST 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.
[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 ketoacid 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 ofα-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,FRST 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.
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