This study addresses the production enhancement of pullulan, an extracellular polysaccharide with various applications. Pullulan is primarily produced by Aureobasidium pullulans (A. pullulans), and genetic modification is commonly used to increase its yield. However, there is a need for a more efficient and safer method. To achieve this, we designed a high-throughput screening system utilizing a unique fluorescent protein specific to pullulan. Ultraviolet (UV) mutagenesis was applied to create a pool of mutant strains, and flow cytometry allowed for single-cell screening. Our approach yielded strain M1-B3, which exhibited a substantial increase in pullulan production from 26.5 g/L to 76.88 g/L. Additionally, the molecular weight of the produced pullulan significantly increased, expanding its potential commercial application. This study demonstrates an efficient and safe method to enhance pullulan production in A. pullulans. The UV mutagenesis and flow cytometry based on screening not only increased yield but also improved pullulan’s molecular weight. The adaptability of this method to other polysaccharides and its potential for genomic analysis and broader applications make it a valuable tool in bioproduction.

L-Lysine, an essential amino acid for humans and mammals, is widely used in the food, feed, medicine, and cosmetics industries. In this study, a lysine over-producing Escherichia coli mutant was isolated using a fluorescence-based screen and an E. coli strain lacking five of the six L-lysine tRNA-UUU genes. Firstly, an l-lysine codon-rich protein was fused with a green fluorescent protein (all AAG codons were replaced with AAA), yielding a rare codon-rich screening marker positively correlated with l-lysine content. After association and room temperature plasma (ARTP) mutagenesis and induced fluorescent protein expression culture, mutant strains with strong fluorescence were sorted using flow cytometry. The fermentation performance of the high-yielding l-lysine strains were evaluated, which resulted in 16 of the 29 mutant strains showing increased L-lysine yields compared with those of the wild-type strains and a screening efficiency of up to 55.2%. Following a 48 h fermentation, the production of l-lysine (14.8 g/L) and biomass by E. coli QD01ΔtRNA L2 were 12.1 and 4.5% higher than those of the wild-type strain. The screening strategy for high-yielding strains based on the artificial rare cryptosystem established in this study will provide an efficient, accurate, and simple method for screening other amino-acid-producing microorganisms.

Aglycone-type soy isoflavones, recognized for their bioactive phytoestrogen properties, face industrial limitations due to their low natural abundance and inefficient conversion. This study optimized a multi-enzyme synergistic catalysis system using soybean sprout powder, achieving high conversion rates and purity through response surface methodology. The optimal enzyme system comprised β-glucosidase (25 U/mL), cellulase (200 U/mL), hemicellulase (400 U/mL), and β-galactosidase (900 U/mL) at pH 5.0, 50 °C, and 3.2 h. This system yielded an aglycone conversion rate of 92% and glycoside hydrolysis rate of 97%, outperforming single-enzyme approaches. Upon post-purification with AB-8 macroporous resin, the product reached a purity of 58.1 ± 0.54% and exhibited strong antioxidant activity, with DPPH and ABTS radical scavenging rates of 81.01 ± 0.78% and 71.37 ± 1.01%, respectively. In a zebrafish central nervous system injury model induced by mycophenolate mofetil, the 500 μg/mL sample group significantly reduced neural apoptosis fluorescence intensity compared to controls (p < 0.05), achieving a neuroprotective rate of 76.58%, which was similar to the effect of L-reducing glutathione. This study offers an efficient, cost-effective enzymatic strategy for producing aglycone soy isoflavones, highlighting their potential in functional foods and neuroprotective applications.

Cellulosome is a highly efficient multi-enzyme self-assembly system and is found on the extracellular surface or in the free environment of microorganisms. However, with a lack of Ca²⁺ in vivo, cellulosome assembly is challenging. In this study, a novel design method was used to directionally modify the Ca²⁺-binding site, and four double-site dockerin A (DocA) mutants were obtained. At a Ca²⁺ concentration between 1.00 × 10⁻⁷ and 1.00 × 10⁻⁴ M, the mutant DocA-D3 had the strongest binding capacity to cohesion (Coh), which was 8.01 times that of DocA. The fluorescence signal intensity of the fusion proteins assembled using mutants was up to 1.26 × 10⁷ in Escherichia coli, which indicated that these mutants could interact with Coh in vivo. The molecular dynamics simulation results showed that DocA-D3 could maintain a stable angle structure without Ca²⁺, and when applied to L-lysine fermentation, the yield was increased by 24.1%; when applied to β-alanine fermentation, the product accumulation was increased by 2.13–2.63 times. These findings lay the foundation for assembly design in cells.

L-leucine, an essential amino acid which cannot be synthesized in mammals, has extensive applications in various fields. However, the large-scale production of L-leucine still faces various challenges in terms of strain and process optimization. In this study, E. coli A211 was used as the initial strain, and a double enhancement strategy of CRISPR-associated transposase genome integration and a plasmid was employed to enhance the L-leucine metabolic pathway. We constructed four engineered strains—E. coli A101, E. coli B201, E. coli CD301, and E. coli bcd401. The transcriptional levels of key genes (leuA, leuCD, leuB, and bcd) in L-leucine biosynthesis were significantly upregulated to boost L-leucine production. Fermentation screening revealed that E. coli CD301 exhibited the highest L-leucine titer (0.57 ± 0.01 g/L), presenting a 97% increase compared with the parental strain. The fermentation process of E. coli CD301 was further optimized using single-factor optimization followed by response surface methodology of variables such as temperature, C/N ratio, and inoculum size, leading to an enhanced L-leucine titer of 0.89 ± 0.03 g/L, a 56.1% improvement over the pre-optimization level. This study demonstrated the effectiveness of CRISPR-associated transposase genome integration and plasmid double enhancement strategy, providing new insights into metabolic engineering approaches for improving L-leucine production via fermentation with E. coli.

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca²⁺, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca²⁺ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca²⁺ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca²⁺ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.