Construction of antibiotic-free riboflavin producer in by metabolic engineering strategies with a plasmid stabilization system.

Riboflavin, an important vitamin utilized in pharmaceutical products and as a feed additive, is mainly produced by metabolically engineered bacterial fermentation. However, the reliance on antibiotics in the production process leads to increased costs and safety risks. To address these challenges, an antibiotic-free riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system.

Continuous Evolution of Protein through T7 RNA Polymerase-Guided Base Editing in .

targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter.

High-Throughput Screening for Enhanced Thermal Stability of Inherently Salt-Tolerant l-Glutaminase and Its Efficient Expression in .

In addressing the challenges posed by extended fermentation cycles and high-salt conditions in high-salt liquid-state fermentation soy sauce (HLFSS) production, a high-throughput screening method was devised to identify thermally stable l-glutaminase mutants. This study yielded mutants A146D and A51D, exhibiting enhanced thermal stability. Computer-aided analysis revealed that these mutations introduced additional forces, compacting the protein structure and lowering the Gibbs free energy, thereby improving thermostability.

The mechanisms of environmental stress tolerance in : progress and perspectives.

have been widely used in industrial compound production for their incomplete oxidation ability. However, they are often subjected to a wide variety of severe environmental stresses, such as extreme pH, high temperature, osmotic pressure, and organic solvents, which greatly repress microbial growth viability and productivity. As typical biocatalysis chassis cells with a high tolerance to external environmental stresses, it is extremely important to construct highly tolerant chassis cells and understand the tolerance mechanisms of and how different stresses interact with the cell: membranes, phospholipid bilayers, transporters, and chaperone proteins.

Multi-module engineering to guide the development of an efficient L-threonine-producing cell factory.

The rapid development of high-productivity strains is fundamental for bio-manufacture in industry. Here, Multi-module metabolic engineering was implemented to reprogram Escherichia coli, enabling it to rapidly transitioning from zero-producer to hyperproducer of L-threonine. Firstly, the synthesis pathway of L-threonine was rationally divided into five modules, and the rapid production of L-threonine was achieved by optimizing the expression of genes in each module.

Engineering for l-Threonine Hyperproduction Based on Multidimensional Optimization Strategies.

Exploring effective remodeling strategies to further improve the productivity of high-yield strains is the goal of biomanufacturing. However, the lack of insight into host-specific metabolic networks prevents timely identification of useful engineering targets. Here, multidimensional engineering strategies were implemented to optimize the global metabolic network for improving l-threonine production.

[Efficient whole-cell biosynthesis of D-mannose by recombinant ].

D-mannose is a natural hexose with great economic and application values in the food, medicine, and cosmetic fields. However, most biosynthesis methods of D-mannose rely on as the host, which poses safety issues during the production process and imposes limitations on subsequent applications. This study compared the enzyme properties of mannose isomerases from multiple sources to select the most suitable source.

[Metabolic engineering of for synthesis of 1,4-butanediol].

1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the synthesis of 1,4-butanediol was achieved after introduction of the path into W3110 (K-12) chassis cells.

[Efficient synthesis of salidroside from tyrosol based on UDPG recycling system].

Salidroside is a functional ingredient with wide applications in food and pharmaceutical fields. It is conventionally produced by extraction from plants, the application of which is limited by the scarcity of raw materials and cumbersome process. This study achieved the efficient production of salidroside by biosynthesis with tyrosol as the substrate.

[Efficient biosynthesis of guanidoacetic acid by a recombinant strain of ].

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of .

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of .

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins.

Comparative transcriptomics analysis-guided metabolic engineering of Yarrowia lipolytica for improved erythritol and fructooligosaccharides production.

Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20-30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times.

Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum.

Application of modern synthetic biology technology in aromatic amino acids and derived compounds biosynthesis.

Aromatic amino acids (AAA) and derived compounds have enormous commercial value with extensive applications in the food, chemical and pharmaceutical fields. Microbial production of AAA and derived compounds is a promising prospect for its environmental friendliness and sustainability. However, low yield and production efficiency remain major challenges for realizing industrial production.

Utilizing 5' UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in .

The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5' UTR sequence from the genome of 168. This sequence resulted in a 5.

Deciphering styrene oxide tolerance mechanisms in Gluconobacter oxydans mutant strain.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses.

Design-build-test of recombinant chassis cell by lifespan engineering for robust bioprocesses.

Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. is widely used in industry for its food safety properties, but challenges remain in the limitations of microbial fermentation. This study proposes a novel strategy based on lifespan engineering to design robust chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis, tolerate toxic substrates, and get a higher mass transfer efficiency.

Microbial production of branched chain amino acids: Advances and perspectives.

Branched-chain amino acids (BCAAs) such as L-valine, L-leucine, and L-isoleucine are widely used in food and feed. To comply with sustainable development goals, commercial production of BCAAs has been completely replaced with microbial fermentation. However, the efficient production of BCAAs by microorganisms remains a serious challenge due to their staggered metabolic networks and cell growth.

N-terminal truncation (N-) and directional proton transfer in an old yellow enzyme enables tunable efficient producing (R)- or (S)-citronellal.

(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of (-)-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production.

Engineering of human tryptophan hydroxylase 2 for efficient synthesis of 5-hydroxytryptophan.

L-Tryptophan hydroxylation catalyzed by tryptophan hydroxylase (TPH) presents a promising method for synthesizing 5-hydroxytryptophan (5-HTP), yet the limited activity of wild-type human TPH2 restricts its application. A high-activity mutant, MT10 (H318E/H323E), was developed through semi-rational active site saturation testing (CAST) of wild-type TPH2, exhibiting a 2.85-fold increase in k/K over the wild type, thus enhancing catalytic efficiency.

Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi.

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae.

Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum.

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering.