L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of Escherichia coli. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the metA-metB-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products.

l-Homoserine is a valuable non-proteinogenic amino acid used in the synthesis of various important compounds. Microbial fermentation has potential value for producing l-homoserine on a large scale, but suffers from a low yield and the need for expensive additives. In this study, a non-induced, non-auxotrophic, plasmid-free Escherichia coli chassis for the high-efficiency production of l-homoserine was constructed. Initially, the l-homoserine degradation pathway was dynamically attenuated. Subsequently, systems metabolic engineering strategies were employed, including reinforcing the synthetic flux, improving NADPH generation, and elevating l-homoserine efflux. The constructed strain HOM-14, produced 60.1 g/L l-homoserine without additional supplements or inducers, which achieved the highest fermentative production efficiency of l-homoserine till date. Moreover, common byproducts, such as acetate, did not accumulate. The strategies presented here can be applied in the further engineering of chassis for the scale-up production of l-homoserine and derivatives.

4–羟基异亮氨酸具有血糖水平依赖的促胰岛素分泌特性,是治疗糖尿病的潜在新药.将异亮氨酸羟化酶(isoleucine dioxygenase,IDO)编码基因ido于大肠杆菌(Escherich coli)中表达,获得工程菌株E.coli pET-ido.以该菌株为研究对象,建立和优化了其合成4–羟基异亮氨酸的静息细胞催化体系.结果表明:在最佳条件下(静息细胞27 g/L,L–异亮氨酸250 mmol/L,α–酮戊二酸250 mmol/L,FeSO48 mmol/L,VC 6 mmol/L,反应温度35℃,pH 9.0)反应12 h,4–羟基异亮氨酸产量和转化率分别达到249.6 mmol/L和99.8％,较优化前均提高了34.5％和7.5％.这是目前已报道的生物法合成4–羟基异亮氨酸的最高产量,为推动该化合物的微生物合成奠定了良好的基础.

为考察组成型过表达异亮氨酸羟化酶(isoleucine dioxygenase,IDO)基因ido对4-羟基异亮氨酸合成的影响,构建ido组成型表达质粒pXM01-ido及菌株HIL017,其ido转录量及IDO活性较诱导型过表达菌株HIL016显著提升,4-羟基异亮氨酸产量较HIL016提高19.4％.为进一步提高4-羟基异亮氨酸产量,通过Plackett-Burman试验确定HIL017发酵培养基中玉米浆、谷氨酸和FeSO4·7H2O用量为主要影响因素,利用最陡爬坡试验和响应面法确定其最优用量为玉米浆34.1 mL/L、谷氨酸2.98 g/L、FeSO4·7H2O 0.0167 g/L,此时4-羟基异亮氨酸理论产量为5.57 g/L.验证实验结果表明,最佳条件下4-羟基异亮氨酸产量为5.53 g/L,较优化前提高19.7％.

苏氨酸是重要的饲料氨基酸,需求量持续增加,提高苏氨酸发酵产率和糖酸转化率,降低生产成本已成为一个重要课题.该实验以苏氨酸工程菌Escherichia coli THRD为出发菌,利用CRISPRi(clustered regularly interspaced short palindromic repeats interference)技术研究中心代谢9个基因转录水平的改变对苏氨酸合成的影响.发酵结果显示,干扰zwf、pfkA和gltA基因的转录水平提高了苏氨酸的合成效率,对应菌株苏氨酸产量分别为60.3、64.6和65.8 g/L,与出发菌(50.9 g/L)相比,分别提高了18.5％、26.9％和29.3％.糖酸转化率分别为40％、38％和39％,与出发菌(34％)相比,分别提高了17.7％、11.8％和14.7％.结果表明,通过CRISPRi干扰中心代谢基因的转录水平,可以调节合成代谢网络,使更多碳源流向苏氨酸,提高苏氨酸的合成效率.同时,该研究也为其他生物制品工程菌的构建提供了参考.

4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5.12 g/L but a considerable amount of L-isoleucine still accumulated in the culture. We then dynamically modulated the activity of the α-ketoglutarate dehydrogenase complex (ODHC) by employing L-isoleucine-responsive transcription or attenuation strategies. The best-engineered strain, HIL18, produced 34.21 g/L 4-HIL with a negligible accumulation of byproducts, including approximately 0.6 g/L L-isoleucine. This study achieved the highest production and yield of 4-HIL, and optimizing the TCA cycle by dynamically modulating the activity of ODHA can be a powerful strategy to balance the carbon flux and achieve efficient production of α-ketoglutarate and derivatives.