Systems Metabolic Engineering for Highly Efficient L-isoleucine Production in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2025-0695

Abstract

Abstract:

Objective L-isoleucine, an essential amino acid, is widely used in pharmaceutical, food, and agricultural industries. To address the limitations of the current industrial strains used for scale-up production of L-isoleucine, such as low synthesis efficiency, prolonged fermentation period, and instability, this study is aimed to construct a highly efficient L-isoleucine-producing strain through systems metabolic engineering. Method ISO-2, an L-isoleucine -producing strain developed previously, was used as the parental strain. Multiple strategies were implemented to enhance its L-isoleucine biosynthesis: Strengthening oxaloacetate and L-aspartate supply, alleviating feedback inhibition of L-isoleucine, enhancing metabolic flux for L-isoleucine synthesis, balancing cofactor levels, introducing citramalate pathway, and reinforcing L-isoleucine efflux. Result The overexpression of ppc, pycA, aspC, and aspA significantly improved oxaloacetate and L-aspartate supply, achieving L-isoleucine titer of 6.98 g/L in strain YL-4. The co-expression of feedback-resistant threonine dehydratase encoding gene ilvA^YI and ilvD, together with NADH-dependent enzymes encoding genes (ilvC^EM and bcd) increased L-isoleucine production by 35.4% in strain YL-8. Implementing the citramalate pathway by cimA and leuBCD elevated L-isoleucine production to 11.03 g/L, and reduced L-valine accumulation to 0.10 g/L in strain YL-12. Knocking out iclR to activate glyoxylate cycle and dynamically regulating sucAB (encoding α-ketoglutarate dehydrogenase) using the auto-regulatory promoter P _fliA boosted L-isoleucine production to 11.93 g/L. Finally, by deleting brnQ (encoding L-isoleucine transporter) and overexpressing ygaZH (encoding L-isoleucine exporter), strain YL-16 produced 49.73 g/L L-isoleucine, with a yield of 0.33 g/g glucose. Conclusion An L-isoleucine hyperproducer with enhanced efficiency and shortened fermentation period was successfully developed via systems metabolic engineering. This combinatorial strategy provides a valuable reference for engineering strains for producing aspartate-family and branched-chain amino acids.

Key words: L-isoleucine, Escherichia coli, metabolic engineering, metabolic flux, dynamic regulation

WEI Min-hua, LI Xiao-tong, JIANG Ya-wen, ZHOU Piao-piao, WANG Kai, SUN Hao, LU Nan, ZHANG Cheng-lin. Systems Metabolic Engineering for Highly Efficient L-isoleucine Production in Escherichia coli[J]. Biotechnology Bulletin, 2025, 41(11): 110-120.

Figures/Tables 14

Table 1 Recent advances in metabolic engineering for L-isoleucine production

底盘细胞 Chassis	主要策略 Main strategies	产量 Titer （g/L）	转化率 Yield （g/g Glucose）	周期 Period（h）	发酵强度Productivity（g/L/h）	参考文献 Reference
谷氨酸棒杆菌 Corynebacterium glutamicum	敲除alaT、brnQ和alr；过表达ilvBN^M、ppnK、lrp、brnFE和ilvA^M	32.1	0.18	72	0.45	［11］
大肠杆菌 Escherichia coli	过表达ilvA^fbr、ilvIH、thrABC、ygaZH、ilvEDA和ilvC	9.5	0.14	60	0.16	［4］
	过表达ilvIH1、ygaZH和CgilvA1；敲除poxB、pflB、ldhA、adhE和tdcC	49.3	0.32	48	1.03	［13］
	敲除brnQ、livj、livk和ackA；过表达ygaZH、cimA3.7、GsleuCD、AfleuB ilvIH^fbr、ilvC^NADH、ilvD、LsleuDH和dcuD；	56.6	0.21	34	1.66	［14］
	过表达ilvA^YI、ilvBN^YI、thrA^fbrBC、pntAB、aspA、brnFE、bcd、metA^G189C和metB^M；敲除brnQ	51.5	0.29	44	1.13	［15］
	过表达thrA^fbrBC、ilvGM（AT）、asd、aspA、ilvEDA^fbr、rhtC和pntAB；敲除pykF和ptsG	5.42	0.40	12	0.83	［16］

Fig. 1 Pathway for synthesis of L-isoleucine and main strategies used in this study

Table 2 Strains and plasmids

菌株/质粒 Strains/Plasmids	特性 Characteristics	来源 Sources
菌株
E. coli DH5α	F-， Δ（lacZYA-argF）U169 recA1endA1 hsdR17	实验室保存
ISO-2	E. coli W3110 ΔlacI P _thrABC -thrA：：P _trc -thrA^fbrylbE：：P _trc -thrA^fbrBC-T _trcyjiP：：P _trc -ilvA^YI-T _trcyncI：：P _trc -ilvBN^YI-T _trc	［15］
YL-1	ISO-2 tehB：：P _trc -ppc	本研究
YL-2	YL-1 yciQ：：P _trc -pycA	本研究
YL-3	YL-2 yeeP：：P _trc -aspC	本研究
YL-4	YL-3 ygaY：：P _trc -aspA	本研究
YL-5	YL-4 yjgX：：P _trc -ilvA^YI	本研究
YL-6	YL-5 yghX：：P _trc -ilvD	本研究
YL-7	YL-6 P _ilvC -ilvC：：P _trc -ilvC^EM	本研究
YL-8	YL-7 P _ilvE -ilvE：：P _trc -bcd	本研究
YL-9	YL-8 yeeL：：P _tr -leuBCD	本研究
YL-10	YL-9 gapC：：P_{BBa_J23110}-cimA	本研究
YL-11	YL-9 gapC：：P_{BBa_J23104}-cimA	本研究
YL-12	YL-9 gapC：：P_{BBa_J23119}-cimA	本研究
YL-13	YL-12 △iclR	本研究
YL-14	YL-13 P _sucAB ：：P _fliA	本研究
YL-15	YL-14 △brnQ	本研究
YL-16	YL-15 yjiV：：P _trc -ygaZH	本研究
质粒
pREDCas9	Spe^R，Cas9和λ Red表达质粒	［17］
pGRB	Amp^R，gRNA表达质粒	［17］
pGRB-tehB	pGRB含tehB靶点sgRNA	本研究
pGRB-yciQ	pGRB含yciQ靶点sgRNA	本研究
pGRB-yeeP	pGRB含yeeP靶点sgRNA	本研究
pGRB-ygaY	pGRB含ygaY靶点sgRNA	本研究
pGRB-yjgX	pGRB含yjgX靶点sgRNA	本研究
pGRB-yghX	pGRB含yghX靶点sgRNA	本研究
pGRB-ilvC	pGRB含ilvC靶点sgRNA	本研究
pGRB-ilvE	pGRB含ilvE靶点sgRNA	本研究
pGRB-yeeL	pGRB含yeeL靶点sgRNA	本研究
pGRB-gapC	pGRB含gapC靶点sgRNA	本研究
pGRB-iclR	pGRB含iclR靶点sgRNA	本研究
pGRB-P _sucAB	pGRB含P _sucAB 靶点sgRNA	本研究
pGRB-brnQ	pGRB含brnQ靶点sgRNA	本研究
pGRB-yjiV	pGRB含yjiV靶点sgRNA	本研究

Fig. 2 Identification maps of YL-1, YL-2, YL-3 and YL-4A-D: Identification maps of YL-1, YL-2, YL-3 and YL-4. M: DNA marker (10 000, 8 000, 7 000, 6 000, 5 000, 40 00, 3 500, 3 000, 25 00, 2 000, 1 500, 1 000, 750, 500, 250 bp); 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlapped PCR product; 5: PCR product amplified from genomic DNA of YL-1, YL-2, YL-3 and YL-4, respectively; 6: PCR product amplified from genomic DNA of the starting strain

Fig. 3 Effects of reprogramming the phosphoenolpyruvate-pyruvate-oxaloacetate-aspartate node on strains' performances

Fig. 4 Identification maps of YL-5-YL-8A-D: Identification maps of YL-5, YL-6, YL-7 and YL-8. M: DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlap PCR product; 5: PCR product amplified from genomic DNA of YL-5, YL-6, YL-7 and YL-8, respectively; 6: PCR product amplified from genomic DNA of the starting strain

Fig. 5 Effects of enhancing L-isoleucine metabolic flux on strains' performances

Fig. 6 Identification maps of YL-9-YL-12A-D: Identification maps of YL-9, YL-10, YL-11 and YL-12; M: DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlap PCR product; 5: PCR product amplified from genomic DNA of YL-9, YL-10, YL-11 and YL-12, respectively; 6: PCR product amplified from genomic DNA of the starting strain

Fig. 7 Effects of introducing the citramalate pathway on strains' performancesA: L-Isoleucine production and biomass of strain YL-9, YL-10, YL-11, and YL-12. B: Intracellular α-ketobutyrate concentration in strain YL-8, YL-9, YL-10, YL-11, and YL-12

Fig. 8 Identification maps of YL-13 and YL-14A: Identification map of YL-13; M: DNA marker; 1: upstream homology arm U iclR; 2: downstream homology arm D iclR; 3: U iclR -D iclR; 4: PCR product amplified from YL-13 genomic DNA; 5: PCR product from YL-12 genomic DNA. B: Identification map of YL-14; M: DNA marker; 1: upstream homology arm UPsucAB; 2: downstream homology arm DPsucAB; 3: P fliA; 4: UPsucAB -P fliA -DPsucAB overlap PCR product; 5: PCR product amplified from YL-14 genomic DNA; 6: PCR product from YL-13 genomic DNA

Fig. 9 Effects of activating glyoxylate cycle and dynamically attenuating TCA cycle on strains' performancesA: α-Ketoglutarate dehydrogenase activity in strain YL-14. B: Production performances of YL-13 and YL-14

Fig. 10 Identification maps of YL-15 and YL-16A: Identification map of YL-15; M: DNA marker; 1: upstream homology arm U brnQ; 2: downstream homology arm D brnQ; 3: U brnQ -D brnQ; 4: PCR product amplified from YL-15 genomic DNA; 5: PCR product from YL-14 genomic DNA. B: Identification map of YL-16; M: DNA marker; 1: upstream homology arm U yjiV; 2: downstream homology arm D yjiV; 3: P trc -ygaZH-T trc; 4: U yjiV -P trc -ygaZH-T trc -D yjiV; 5: PCR product amplified from YL-16 genomic DNA; 6: PCR product amplified from YL-15 genomic DNA

Fig. 11 Effects of modifying L-isoleucine transportation system on production performancesA: Fermatention performances of YL-15 and YL-16. B: Intracellular L-isoleucine levels in YL-14, YL-15, and YL-16

Fig. 12 Fed-batch fermentation process curve of YL-16 in a 5 L bioreactor

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