Advances in bioproduction of feed amino acid by Escherichia coli

doi:10.12211/2096-8280.2021-042

Abstract

Abstract:

With the rapid development of animal husbandry and fishery, it promotes the development of protein for animal feed. Due to the enhancement of people's safety awareness of food additives, there is an urgent need to enhance the supply of sustainable protein for use in animal feed. Because the protein is composed of amino acids, the feed amino acids can replace protein for animal feed, which can provide enough nutrition for the animal's growth. Thus, the feed amino acid is an important product that has attracted a great attention by investigators because of the widely uses in the fields of animal feed supplement and has broad market potential. Using synthetic biology and metabolic engineering strategy, the metabolic pathway and regulatory network of Escherichia coli (E. coli) was designed, engineered, and reconstructed to obtain suitable E. coli cell factories.These factories provide a promising and sustainable alternative for the production of feed amino acid from renewable feedstock, and is attracting great attention as it is an economic and environmentally friendly bioprocessing. E. coli cell factories offer an alternative strategy to replace chemical refinery, animal and plant extraction, reducing the harm to the environment and dependence on natural resources. In the present review, we discussed the biosynthesis pathway of feed amino acid (lysine, methionine, tryptophan, threonine, valine, and arginine) in E. coli. According to the biosynthesis pathway of feed amino acid, the production bottlenecks of feed amino acid in E. coli cell factories were discussed. We also summarized recent studies about in the feed amino acid production from the constructed and optimized of E. coli cell factory. Furthermore, we proposed future research directions to improve the technical level of feed amino acid and enhance the robustness of E. coli cell factories for reducing the cost of bioproduction, simplifying downstream separation, and enhancing industrial productivity.

Key words: feed amino acid, Escherichia coli, microbial cell factories, synthetic biology, metabolic engineering

摘要：

随着畜牧业的快速发展，人们对畜牧饲料蛋白的需求日益剧增。由于人们对食品安全意识的增强，迫切需要开发安全、高效、可持续动物饲料蛋白的供应途径。由于氨基酸是组成蛋白质的基本单元，所以在饲料中添加氨基酸可以替代饲料中的蛋白质，为动物细胞生长发育提供足够的营养。因此，饲用氨基酸作为动物饲料食品添加剂被广泛应用，具有广阔的市场应用前景。利用合成生物学技术，工程化改造大肠杆菌，构建的细胞工厂，以生物质为原料可绿色高效合成饲用氨基酸，而且其具有原料可再生、成本低廉、反应条件温和、环境污染小等优点，为解决动植物提取和化学炼制引起的环境污染问题提供了一种有效解决方案。本文针对饲用氨基酸（赖氨酸、甲硫氨酸、色氨酸、苏氨酸、缬氨酸和精氨酸）的生物合成途径，介绍了大肠杆菌合成饲用氨基酸的生产瓶颈，并从饲用氨基酸大肠杆菌细胞工厂的构建与优化，综述了利用合成生物学技术改造大肠杆菌细胞工厂合成饲用氨基酸的研究现状。提升饲用氨基酸的生产技术水平、提高大肠杆菌细胞的鲁棒性和增强大肠杆菌细胞对不利环境的耐受能力，可以提升饲用氨基酸发酵性能，简化发酵过程控制，降低饲用氨基酸的生产成本，是未来饲用氨基酸生产菌株工程化改造的方向。

关键词: 饲用氨基酸, 大肠杆菌, 微生物细胞工厂, 合成生物学, 代谢工程

CLC Number:

Liang GUO, Cong GAO, Yadi LIU, Xiulai CHEN, Liming LIU. Advances in bioproduction of feed amino acid by Escherichia coli[J]. Synthetic Biology Journal, 2021, 2(6): 964-981.

郭亮, 高聪, 柳亚迪, 陈修来, 刘立明. 大肠杆菌生产饲用氨基酸的研究进展[J]. 合成生物学, 2021, 2(6): 964-981.

Figures/Tables 5

Fig. 1 Metabolic pathways of aspartate family amino acids and feedback regulations involved in E. coli(Blue dotted lines indicate feedback repression, red dotted lines indicate feedback inhibition, and solid arrows indicate one-step metabolic pathways)Key enzymes in metabolic pathway: AAT—aspartate aminotransferase; AK Ⅰ, AK Ⅱ, AK Ⅲ—aspartate kinase; ASD—aspartate semialdehyde dehydrogenase; HD Ⅰ, HD Ⅱ—homoserine dehydrogenase; HK—homoserine kinase; TS—threonine synthase; DS—dihydrodipicolinate synthase; DR—dihydrodipicolinate reductase; THS—tetrahydrodipicolinate succinyltransferase; SDAT Ⅰ, SDAT Ⅱ—succinyl diaminopimelate (DAP) aminotransferase; SDD—succinyl DAP desuccinylase; DE—L,L-DAP epimerase; DDC—meso-DAP decarboxylase; HST—homoserine succinyltransferase; SHL—succinyl homoserine lyase; CL—cystathionine lyase; HMT Ⅰ, HMT Ⅱ—homocysteine methyltransferase.Key genes in metabolic pathway: aspC—aspartate aminotransferase-encoding gene; thrA—aspartate kinase Ⅰ-encoding gene; metL—aspartate kinase Ⅱ-encoding gene; lysC—aspartate kinase Ⅲ-encoding gene; asd—aspartate semialdehyde dehydrogenase-encoding gene; thrA—homoserine dehydrogenase Ⅰ-encoding gene; thrB—homoserine kinase-encoding gene; thrC—threonine synthase-encoding gene; dapA—dihydrodipicolinate synthase-encoding gene; dapB—dihydrodipicolinate reductase-encoding gene; dapD—tetrahydrodipicolinate succinyltransferase-encoding gene; argD—succinyl diaminopimelate (DAP) aminotransferase Ⅰ-encoding gene; argM—succinyl diaminopimelate (DAP) aminotransferase Ⅱ-encoding gene; dapE—succinyl DAP desuccinylase-encoding gene; dapF—L,L-DAP epimerase; lysA—meso-DAP decarboxylase-encoding gene; metA—homoserine succinyltransferase-encoding gene; metB—succinyl homoserine lyase-encoding gene; metC—cystathionine lyase-encoding gene; metE—homocysteine methyltransferase Ⅰ-encoding gene; metH—homocysteine methyltransferase Ⅱ-encoding gene; metK—methionine adenosyltransferase-encoding gene; gdh—glutamate dehydrogenase-encoding gene; glyA—serine hydroxymethyltransferase-encoding gene; metF—methylenetetrahydrofolate reductase-encoding gene)

Tab. 1 Comparison of feed amino acid production by E. coli cell factories

产品	菌株	性状	发酵方式	产量 /(g/L)	生产强度 /[g/(L·h)]	文献
赖氨酸	E. coli NT1003	E. coli Δmet Δthr ↑ppc ↑pntB ↑aspA	发酵罐	134.9	1.87	[47]
	E. coli LATR11/pWG-DC^SMA^SMBH_c.gLP	E. coli LATR11 ↑lysC^T344M ↑asd ↑dapA^H56K↑dapB ↑lysA ↑ppc ↑ddh	发酵罐	125.6	3.14	[48]
	E. coli DL2	E. coli MG1655 ↑lysC^T253R ↑dapA^E84T	摇瓶	9.5	—	[49]
	E. coli RS3	E. coli DL2 ↑lysC^D340P ↑dapA^E84TspeB^A302VatpB^S165NsecY^M145V	发酵罐	155	3.69	[50]
	E. coli Lc_(H)‐Fe_(M)	E. coli CCTCC M2019435 ↑lysC ↑fre	发酵罐	193.6	4.03	[53]
苏氨酸	E. coli TWF006/pFW01-thrA^*BC-asd	E. coli TWF006 ↑thrBC ↑asd ↑thrA^S345A	摇瓶	15.85	0.44	[41]
	E. coli W3110 pWYE134	E. coli W3110 ↑thrL ↑thrBC ↑thrA^S345A	发酵罐	9.22	0.19	[54]
	E. coli THPZ	E. coli THRD ↑zwf	发酵罐	126.1	5.25	[55]
	E. coli TWF106/pFT24rp	E. coli TWF001 ΔpoxB ΔpflB ΔldhA ΔadhE ΔtdcC ↑rhtC ↑pycmt	发酵罐	80	2.22	[36]
	E. coli THPE5	E. coli THRD ↑pycA ↑pckA ↑fdh ↑aspC↑gdhA ↑udhA ↑citA	发酵罐	70.8	1.77	[56]
甲硫氨酸	E. coli W3110 ΔmetJ/pTrcA*H	E. coli W3110 ΔmetJ ΔmetA ΔlysA ↑yjeH	发酵罐	9.75	0.20	[38]
	E. coli M3/PAmZ	E. coli W3110 IJAHFEBC ↑metA^fbr ↑yjeH↑serA^fbr ↑metZ	摇瓶	3.96	0.083	[57]
	E. coli Me05 (pETMA^Fbr-B-Y/pKKmetH)	E. coli W3110 ΔmetJ ΔthrC Δ lysA ↓metKp^G↑metA^Fbr ↑metB ↑metH	发酵罐	5.62	0.12	[58]
	E. coli W3110 IJAHFEBC/pAm	E. coli W3110 IJAHFEBC ↑metA^Fbr ↑yjeH↑serA^fbr	发酵罐	16.86	0.35	[39]
色氨酸	E. coli w3110 trpAE1 trpR tnaA	E. coli W3110 ΔtrpR ΔtnaA ↑trpEDCBA	发酵罐	54.5	0.70	[59]
		E. coli FB-04/pSV03E. coli W3110 ΔtrpRΔtnaA ΔpheA ΔtyrA ↑aroF^fbr ↑trpE^S40FD	发酵罐	13.3	0.029	[60]
	E. coli Tna (pSC101 trp·I15)	—	发酵罐	6.2	0.23	[61]
	E. coli KW023	E. coli KW ΔtrpR ΔpykF ΔptsH ↑trpEDCBA↑aroG^fbr ↓pta ↑galP ↑glk	发酵罐	39.7	1.6	[62]
	E. coli T16	E. coli TRTH Δppc ↑acs ↑aceB ↑mdh ↑pck	发酵罐	54.6	1.52	[63]
缬氨酸	E. coli Val (pKBRilvBNCED, pTrc184ygaZHlrp	E. coli Val ↑ilvBNCED ↑ygaZH ↑lrp	摇瓶	7.55	0.16	[64]
	E. coli VAMF (pKBRilvBN^mutCED, pTrc184ygaZHlrp)	E. coli VAMF ↑ygaZH ↑lrp ↑ilv ↑ilvBN^mutCED	发酵罐	32.3	0.58	[65]
	E. coli V1 cat-PL-ilvE5.7	E. coli K12 ΔilvIH ΔilvBN ΔlvGME ↑ilvE ↑ilvBN^N17KDA	摇瓶	9.8	0.20	[66]
	E. coli VHY18	W3110 ΔlacI ΔycdN ΔycgH ΔydeU ΔyjiT Δyee ΔyjiV ΔpflB ΔldhA ΔadhE↑alsS ↑bcd ↑ilvD↑ilvCM ↑brnFE ↑spoT^[^{R290E, K292D}^]	发酵罐	84	2.33	[67]
精氨酸	E. coli JM109-9039	E. coli JM109 ΔargR ↑argCJBDFRGH	摇瓶	0.040	—	[68]
	E. coli SJB009	E. coli C600⁺ ΔspeC ΔspeF ΔadiA ΔargR ΔargA ↑argA215 ↑argO	发酵罐	11.64	0.24	[69]
	E. coli KO	E. coli MG1655 ↑argO ↑argAH15A ΔargR	摇瓶	1.03	—	[70]

Tab. 1 Comparison of feed amino acid production by E. coli cell factories

产品	菌株	性状	发酵方式	产量 /(g/L)	生产强度 /[g/(L·h)]	文献
赖氨酸	E. coli NT1003	E. coli Δmet Δthr ↑ppc ↑pntB ↑aspA	发酵罐	134.9	1.87	[47]
	E. coli LATR11/pWG-DC^SMA^SMBH_c.gLP	E. coli LATR11 ↑lysC^T344M ↑asd ↑dapA^H56K↑dapB ↑lysA ↑ppc ↑ddh	发酵罐	125.6	3.14	[48]
	E. coli DL2	E. coli MG1655 ↑lysC^T253R ↑dapA^E84T	摇瓶	9.5	—	[49]
	E. coli RS3	E. coli DL2 ↑lysC^D340P ↑dapA^E84TspeB^A302VatpB^S165NsecY^M145V	发酵罐	155	3.69	[50]
	E. coli Lc_(H)‐Fe_(M)	E. coli CCTCC M2019435 ↑lysC ↑fre	发酵罐	193.6	4.03	[53]
苏氨酸	E. coli TWF006/pFW01-thrA^*BC-asd	E. coli TWF006 ↑thrBC ↑asd ↑thrA^S345A	摇瓶	15.85	0.44	[41]
	E. coli W3110 pWYE134	E. coli W3110 ↑thrL ↑thrBC ↑thrA^S345A	发酵罐	9.22	0.19	[54]
	E. coli THPZ	E. coli THRD ↑zwf	发酵罐	126.1	5.25	[55]
	E. coli TWF106/pFT24rp	E. coli TWF001 ΔpoxB ΔpflB ΔldhA ΔadhE ΔtdcC ↑rhtC ↑pycmt	发酵罐	80	2.22	[36]
	E. coli THPE5	E. coli THRD ↑pycA ↑pckA ↑fdh ↑aspC↑gdhA ↑udhA ↑citA	发酵罐	70.8	1.77	[56]
甲硫氨酸	E. coli W3110 ΔmetJ/pTrcA*H	E. coli W3110 ΔmetJ ΔmetA ΔlysA ↑yjeH	发酵罐	9.75	0.20	[38]
	E. coli M3/PAmZ	E. coli W3110 IJAHFEBC ↑metA^fbr ↑yjeH↑serA^fbr ↑metZ	摇瓶	3.96	0.083	[57]
	E. coli Me05 (pETMA^Fbr-B-Y/pKKmetH)	E. coli W3110 ΔmetJ ΔthrC Δ lysA ↓metKp^G↑metA^Fbr ↑metB ↑metH	发酵罐	5.62	0.12	[58]
	E. coli W3110 IJAHFEBC/pAm	E. coli W3110 IJAHFEBC ↑metA^Fbr ↑yjeH↑serA^fbr	发酵罐	16.86	0.35	[39]
色氨酸	E. coli w3110 trpAE1 trpR tnaA	E. coli W3110 ΔtrpR ΔtnaA ↑trpEDCBA	发酵罐	54.5	0.70	[59]
		E. coli FB-04/pSV03E. coli W3110 ΔtrpRΔtnaA ΔpheA ΔtyrA ↑aroF^fbr ↑trpE^S40FD	发酵罐	13.3	0.029	[60]
	E. coli Tna (pSC101 trp·I15)	—	发酵罐	6.2	0.23	[61]
	E. coli KW023	E. coli KW ΔtrpR ΔpykF ΔptsH ↑trpEDCBA↑aroG^fbr ↓pta ↑galP ↑glk	发酵罐	39.7	1.6	[62]
	E. coli T16	E. coli TRTH Δppc ↑acs ↑aceB ↑mdh ↑pck	发酵罐	54.6	1.52	[63]
缬氨酸	E. coli Val (pKBRilvBNCED, pTrc184ygaZHlrp	E. coli Val ↑ilvBNCED ↑ygaZH ↑lrp	摇瓶	7.55	0.16	[64]
	E. coli VAMF (pKBRilvBN^mutCED, pTrc184ygaZHlrp)	E. coli VAMF ↑ygaZH ↑lrp ↑ilv ↑ilvBN^mutCED	发酵罐	32.3	0.58	[65]
	E. coli V1 cat-PL-ilvE5.7	E. coli K12 ΔilvIH ΔilvBN ΔlvGME ↑ilvE ↑ilvBN^N17KDA	摇瓶	9.8	0.20	[66]
	E. coli VHY18	W3110 ΔlacI ΔycdN ΔycgH ΔydeU ΔyjiT Δyee ΔyjiV ΔpflB ΔldhA ΔadhE↑alsS ↑bcd ↑ilvD↑ilvCM ↑brnFE ↑spoT^[^{R290E, K292D}^]	发酵罐	84	2.33	[67]
精氨酸	E. coli JM109-9039	E. coli JM109 ΔargR ↑argCJBDFRGH	摇瓶	0.040	—	[68]
	E. coli SJB009	E. coli C600⁺ ΔspeC ΔspeF ΔadiA ΔargR ΔargA ↑argA215 ↑argO	发酵罐	11.64	0.24	[69]
	E. coli KO	E. coli MG1655 ↑argO ↑argAH15A ΔargR	摇瓶	1.03	—	[70]

Fig. 2 Schematic of the tryptophan biosynthetic pathway in E. coliKey enzymes in metabolic pathway: PykF, PykA—pyruvate kinase; PpsA—phosphoenolpyruvate synthase; AroG—3-deoxy-D-arabinoheptulosonate-7-phosphate (DHAP) synthase; AroB—3-dehydroquinate synthase; AroD—3-dehydroshikimate dehydratase; AroE—shikimate 5-dehydrogenase; AroK—shikimate kinase; AroA—EPSP synthase; AroC—chorismate synthase; TrpE—anthranilate synthase I; TrpD—anthranilate synthase II; TrpC—(1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate isomerase; TrpB—tryptophan synthase B; TrpA—tryptophan synthase A; TyrA—chorismate dehydrogenase; PheA—chorismate dehydratase

Fig. 3 Schematic of valine biosynthetic pathway in E. coli (Green dotted arrows indicate feedback inhibition by valine, blue dotted arrows indicate feedback repression, red arrows indicate activation of gene expression, and black arrows indicate one-step metabolic pathways)Key enzymes in metabolic pathway: IlvC—acetohydroxy acid isomeroreductase; IlvD—dihydroxy acid dehydratease; Bcd—leucine dehydrogenase; LeuA—isopropylmalate synthase; LeuCD—isopropylmalate isomerase; LeuB—3-isopropylmalate dehydrogenase; ThrB, IlvE—aromatic amino acid transaminase; PanB—3-methyl-2-oxobutanoate hydroxymethyltransferase; PanE—2-dehydropantoate reductase; PanC—pantoate-β-alanine ligase Key genes in metabolic pathway: livJ—valine transporter-encoding gene; ygaZH—valine exporter-encoding gene; ilvBN—acetohydroxy acid synthase I-encoding gene; ilvGM—acetohydroxy acid synthase II-encoding gene; ilvIH—acetohydroxy acid synthase Ⅲ-encoding gene

Fig. 4 Schematic of arginine biosynthetic pathway inmicroorganismsKey enzymes in metabolic pathway:ArgA—acetylglutamate synthase; ArgJ—ornithine acetyltransferase; ArgB—acetylglutamate kinase;ArgC—acetylglutamate semialdehyde dehydrogenase; ArgD—acetylornithine transaminase; ArgE—acetylornithine deacetylase; ArgF—ornithine transcarbamylase; ArgF′—acetylornithine carbamoyltransferase; ArgG—argininosuccinate synthase; ArgH—arginosuccinase

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