Jul 2026· Journal of Animal Science and Biotechnology· Vol 17· 0 citations· 50 references
Medicine
Abstract
L-Isoleucine is an essential branched-chain amino acid for livestock and poultry, supporting protein accretion and regulating energy metabolism, immune function, and stress resilience. A genetically stable L-isoleucine producer, Corynebacterium glutamicum cgl-Ile0, was obtained via biosensor-assisted ARTP mutagenesis, and produced 11.52 g/L L-isoleucine in a 5-L fermenter, with a yield of 0.11 g/g and a productivity of 0.24 g/L/h. Whole-genome resequencing revealed four mutations associated with the phenotype, including aspBV346I, asdP27E, brnEL87S, and brnFR28P. Structure-guided protein engineering of two rate-limiting enzymes—threonine dehydratase (TD) and acetohydroxyacid synthase (AHAS)—generated strain cgl-Ile1, increasing titer, yield, and productivity by 39.50%, 27.27%, and 37.50%, respectively, relative to strain cgl-Ile0. Subsequent modular optimization of L-isoleucine biosynthesis, oxaloacetate supply module, cofactor-supply module, and transport/export module yielded the final strain cgl-Ile8. Through optimization pH and dissolved oxygen, the titer, yield and productivity of L-isoleucine produced by strain cgl-Ile8 in a 5-L fermenter were 48.49 g/L, 0.31 g/g and 1.01 g/L/h, which were 4.21-, 2.82-, and 4.21-fold those of strain cgl-Ile0, respectively. Scale-up to a 50-L fermenter further increased the titer, yield and productivity to 50.12 g/L, 0.32 g/g and 1.04 g/L/h, respectively, representing the highest reported L-isoleucine titer in C. glutamicum to date. We developed an industrial L-isoleucine-producing C. glutamicum strain by integrating biosensor-guided ARTP mutagenesis, structure-guided protein engineering, and modular pathway rewiring, providing a practical and transferable framework for constructing GRAS amino-acid producers for animal nutrition.
This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.
Qing Yang, Tao-Shun Zhou, Bo Zhang et al.· Biotechnology and Bioenginee...· 0 citations
Branched-chain amino acids (BCAAs), comprising L-valine, L-leucine, and L-isoleucine, are essential amino acids with extensive applications in food, feed, pharmaceuticals, and cosmetics. Escherichia coli and Corynebacterium glutamicum, the two predominant industrial workhorses have been extensively engineered for high-level BCAAs biosynthesis. This review presents a comprehensive analysis of recent advances in microbial BCAAs production through a Mechanism-Module-Process framework. From the mechanism dimension, the intricate BCAA biosynthetic architectures are delineated, encompassing allosteric feedback inhibition, transcriptional attenuation, and transport system. From the module dimension, modular metabolic engineering strategies are dissected, including precursor supply enhancement, NADPH cofactor rebalancing, biosensor-driven dynamic regulation, and adaptive laboratory evolution for strain robustness. From the process dimension, how oxygen availability reprograms cellular metabolism across aerobic, microaerobic, and anaerobic fermentation is systematically examined, and how two-stage fermentation coupled with cofactor rebalancing resolves the growth-production trade-off to achieve near-theoretical yields is discussed. Furthermore, the biosynthetic strategies and current production status of BCAAs derivatives are summarized, highlighting the importance of multidimensional framework engineering. Collectively, this Mechanism-Module-Process framework provides a holistic roadmap for constructing superior BCAAs cell factories and extending their metabolic potential toward derivative biosynthesis.
Guihong Zhao, Si-Yu Tian, Jiong-Ran Li et al.· Biotechnology Advances· 0 citations
This study demonstrates the combined optimization of isozyme combination and environmental stress to elevate the synthesis of astaxanthin and other carotenoids in D. salina, providing new research ideas and experimental evidence for the future construction of high-yield engineered algal strains.
Hao Zhang, Yifan Kong, Yaping Shao et al.· World Journal of Microbiolog...· 0 citations
As a high-value-added amino acid derivative, trans-4-hydroxy-L-proline (T-4-Hyp) faces key bottlenecks in its microbial production from glucose, including insufficient precursor supply and an imbalance between cell growth and product biosynthesis. In this study, we successfully constructed an engineered Escherichia coli strain QF-27 for efficient T-4-Hyp production. First, the L-proline (L-Pro) biosynthetic pathway was enhanced by overexpressing the feedback-resistant γ-glutamyl kinase, glutamate-γ-semialdehyde dehydrogenase, and pyrroline-5-carboxylate reductase, and by knocking out the L-proline dehydrogenase. The resulting strain QF-9 produced 15.75 ± 0.56 g/L of L-Pro. Subsequently, the expression level of proline-4-hydroxylase from Dactylosporangium sp. RH1 was optimized in strain QF-9, and the resulting strain QF-14 produced 5.32 ± 0.26 g/L of T-4-Hyp. To address the insufficient supply of α-ketoglutarate (α-KG), a multi-modular synergistic strategy (i.e., blocking byproduct pathways, enhancing α-KG flux, and relieving global transcriptional repression) increased T-4-Hyp production to 11.02 ± 0.27 g/L. Moreover, a dynamic switch combining PrpsT promoter and DAS+4 degradation tag was designed to repress expression and promote degradation of the α-ketoglutarate dehydrogenase complex during the stationary phase, thereby balancing cell growth and T-4-Hyp production. Consequently, T-4-Hyp production reached 14.37 ± 0.46 g/L, and residual L-Pro fell to 0.38 ± 0.17 g/L. In fed-batch fermentation, the final strain QF-27 produced 105.72 ± 0.84 g/L of T-4-Hyp with productivity of 2.20 g/L/h and carbon yield of 0.364 g/g glucose. To our knowledge, this is the best performance reported for T-4-Hyp production by microbial fermentation, and the first study to enhance it via systematic modification of central carbon metabolism.
Kai Wang, Shu-Ping Tian, Shuo Wan et al.· Metabolic Engineering· 0 citations
S-Adenosylmethionine (SAM) is a high-value biomolecule with critical applications in nutraceuticals, pharmaceuticals, and health supplements. However, the detailed metabolic mechanism by which sodium citrate promotes SAM production in Pichia pastoris has not yet been elucidated. Here, a comprehensive time-series transcriptomic analysis revealed that sodium citrate profoundly influences gene expression across multiple pathways. Sodium citrate supplementation redirects carbon flux toward oxidative energy metabolism by upregulating amino acid biosynthesis, translation, and glycolysis. Crucially, the transcriptional upregulation of argininosuccinate synthase (ARG1) and argininosuccinate lyase (ARG4) was identified as a key node driving fumarate-mediated TCA cycle anaplerosis and ATP supply. This foundational analysis led to the identification of ARG4 as a key metabolic engineering target. Overexpression of ARG4 significantly improved SAM production, achieving a 56.46% increase in shake flasks and 9.06 g/L SAM (a 22.10% improvement) in 500 mL fermenters compared to control strains. Integrated physiological and metabolic flux analysis (MFA) demonstrated that ARG4 overexpression redirects metabolic flow, channeling energy substrates toward SAM synthesis, effectively maintaining cellular respiratory metabolism and alleviating energy limitations in the late fermentation phase. This work establishes a novel strategy for enhancing product synthesis by restructuring energy allocation rather than merely increasing overall energy supply.
l-arginine is widely used in food, feed, pharmaceutical, and cosmetic industries. However, its industrial-scale biosynthesis is limited by insufficient coordination between metabolic regulation, pathway engineering, and fermentation optimization. In this study, an enzyme-constrained model (ec_iML1515) was used to identify 11 gene targets affecting l-arginine production. Based on these targets, metabolic reprogramming was performed in strain Arg4 to rebalance precursor pools (oxaloacetate, aspartate, and citrulline), generating strain Arg10 with an l-arginine titer of 87.24 g/L. Subsequently, the rate-limiting enzyme argininosuccinate synthetase (ArgG) was engineered to the optimal mutant ArgGY131F/K132R and genomically integrated to construct the strain Arg11, increasing the l-arginine titer to 94.80 g/L while reducing aspartate accumulation 7.6-fold to 1.1 g/L. Finally, after the optimization of fermentation temperature and pH, the l-arginine titer, yield, and productivity of strain Arg11 were 114.18 g/L, 0.57 g/g, and 2.27 g/L/h, respectively, in a 3-m3 fermenter, achieving the best performance reported to date.
Shengyang He, Qi Sheng, Gang Men et al.· Bioresource Technology· 0 citations
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