As a new interdisciplinary subject that covers many fields in the 21^st century, synthetic biology is of great help in exploring the basic laws of life activities and biotechnological innovations and breakthroughs. Synthetic biotechnology is a new approach to construct engineered microorganisms. It has been widely used to produce amino acids, organic acids, aromatic compounds, sugars, and so on. The yield of target products produced by recombinant engineered microorganisms can be efficiently increased by using advanced modules, systematic design and new genome editing methods to engineer the complex metabolic pathways in type strains, promoting the rapid development of social productivity. In this paper, the latest advances in the application of synthetic biology for the construction of engineered microorganism are reviewed, the developmental course of synthetic biotechnology is elaborated, and its application for the construction of engineered microorganism is exemplified. With the deepening of research on engineered microorganisms, synthetic biotechnology will bring new breakthroughs and opportunities for the fermentation industry.

In the present study, four key residues in aspartokinase were selected for site-directed saturation mutagenesis, namely, Thr 379 and Ala 380 (the binding sites of the inhibitor Lys), Thr 65 (the binding site of the substrate Asp) and Asp 173 (the catalytically active site of both substrates and ATP binding site). The mutant T379N/A380C/T65I/D173G was successfully constructed using high-throughput screening. Compared with the wild type (WT) enzyme, the activity of the mutant was increased by 75.83 times. In addition, the characterization of kinetics and enzymatic properties showed that the K_m value of the mutant was reduced (1.34 versus 4.11 mmol/L) compared to the WT enzyme, indicating increased substrate affinity. The n value was reduced (1.07 versus 1.71), suggesting that the positive synergistic effect was weakened. Moreover, the optimal temperature, pH and half-life of the mutant were 30 ℃, 8.0 and 3.23 h compared to 25 ℃, 8.0 and 4.24 h for the WT enzyme, respectively. The inhibition effect of the inhibitors at 0.2–10 mmol/L on the mutant was weakened, especially for Lys and Lys + Thr. Through seamless cloning, it was transformed into Corynebacterium pekinense to construct an engineered bacterium. Compared with WT, the yields of lysine, threonine and methionine were increased by 83.05%, 29.36% and 30.77%, respectively. This work will provide a theoretical basis for optimizing the AK metabolic pathway and constructing strains capable of producing aspartate-family amino acids in high yield.

The purpose of this study is to improve the catalytic activity of aspartate kinase (AK), the first key rate-limiting enzyme in aspartic acid metabolism pathway by site-directed random mutagenesis; reduce or remove the synergistic feedback inhibition of metabolites on it; and analyze its spatial structure using Discovery Studio (DS) software and the underlying mechanism through molecular dynamics simulation (MDS). First, the key residue sites around ATP were chosen for further construction of mutants based on the mutant obtained in our previous study, T379N/A380C/G171I. Then, through high-throughput screening, the mutant T379N/A380C/G171I/S227D with significantly increased AK activity was selected from the mutants. Kinetic analysis showed that the V_max value of T379N/A380C/G171I/S227D was 242.05 U/(mg·min), which was 1.28 and 80.41 times higher than that of T379N/A380C/G171I (187.88 U/(mg·min)) and the wild-type (WT) strain (3.01 U/(mg·min)), respectively. The K_m value decreased to 1.35 mmol/L, and the substrate affinity was increased. Through DS software and MDS analysis, it was found that the system became more stable after the mutation, the hydrogen bond occupancy rate with ATP was increased, and the substrate binding stability was enhanced, thereby favoring the catalytic reaction. The enzymatic properties showed that the optimal reaction temperature of T379N/A380C/G171I/S227D was 30 ℃, 5 and 2 ℃ higher than the wild-type strain and T379N/A380C/G171I, respectively; the optimal pH was 8.5 compared to 9.0 for T379N/A380C/G171I; the half-life was 3.9 h, which was 0.8 h longer than T379N/A380C/G171I. In addition, in the presence of different concentrations of the inhibitors Lys, Thr and Met as well as different combinations of Lys + Thr, Lys + Met, Thr + Met and Lys + Thr + Met, the mutant strain was activated by up to 143.35% rather than inhibited.

Acetohydroxy acid synthase (AHAS), a thiamine diphosphate (ThDP)-dependent enzyme, is the key enzyme that catalyzes the first step in the production of branched chain amino acids (BCAA), namely L-valine, L-leucine and L-isoleucine. AHAS is susceptible to feedback inhibition by the end product, BCAA, which inhibits the activity of the enzyme and affects carbon flux to BCAA. Therefore, AHAS is an important target for high-yield production of BCAA, and the modification of AHAS is of great significance. In this review, the structure, catalytic mechanism, and role of AHAS in the BCAA synthesis pathway are introduced. The current research status of the catalytic process of the enzyme and the current molecular modification strategies for AHAS are summarized. Finally, future research directions and modification strategies for the enzyme are proposed.

Site-directed mutagenesis was used to improve the catalytic activity of homoserine dehydrogenase (HSD) to reduce its feedback inhibition and repression by metabolites in the pathway. HSD was docked with the substrate homoserine, and its spatial structure was analyzed. Two key sites, Gly25 and Asp61, were selected for site-directed saturation mutation. The activity screening showed that the mutants A61L and G25G had significantly increased enzyme activity when compared to the wild type (WT). The kinetics and enzymatic properties of these two mutants were studied. It was found that compared to the WT enzyme, the K_m values of G25G and A61L decreased, the substrate affinity increased, and the enzyme activity increased by 1.21 and 1.35 times, respectively; the n value decreased, and the positive synergy increased. The optimum temperature for A61L and G25G was 40 ℃, the same as that for WT; the optimum pH for A61L and WT was 8.0, which was lower than that (8.5) for G25G. The half-lives of A61L and G25G were 1 h longer and 0.5 h shorter than that of WT, respectively. Low concentrations of K⁺, Mg²⁺, and Ca²⁺ could activate the mutants and WT, while different concentrations of methanol, ethanol, acetonitrile and dimethyl sulfoxide had significantly inhibitory effects on the mutants and WT. At inhibitor concentrations of 1–25 mmol/L, the inhibitory effect was significantly weaker on the mutants than on WT. The mutants G25G and A61L showed improved enzyme activity and weakened allosteric inhibition. This study provides a reference for optimizing the biosynthetic pathway of HSD and constructing strains capable of producing high yield of methionine, threonine and isoleucine.

L-Methionine is the only sulfur-containing essential amino acid. It acts as a precursor in the synthesis of various biologically active substances and participates in various metabolic pathways in the body. It is widely used in food, animal feed, medicine, cosmetics, and other fields. In recent years, since L-methionine is the only essential amino acid that cannot be industrially produced by microbial fermentation, the potential of metabolic engineering to improve L-methionine production has received widespread attention from researchers around the world. In this paper, the biosynthesis pathways and metabolic regulation mechanisms of L-methionine in Corynebacterium glutamicum and Escherichia coli are analyzed and compared. The metabolic engineering strategies to produce L-methionine are reviewed from five aspects: the removal of feedback inhibition of key enzymes, the cut-off or weakening of branch metabolic pathways, the optimization of the central metabolic regulatory network, the enhancement of cofactor supply, and the optimization of transport systems, and recent progress in research on the biosynthesis of L-methionine is summarized. Finally, future prospects are also discussed. It is hoped that this review will provide a basis for the breeding of high-yield L-methionine-producing strains.