Aug 2026· Bioresources and Bioprocessing· Vol 13· 0 citations· 38 references
Medicine
Abstract
3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L−1 in shake flasks and 102.18 g L−1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.
Efficient biosynthesis of 1,3-PDO, a key bio-based chemical, depends on precise regulation of the host metabolic network. In this study, a heterologous CRISPR-Cas12a genome editing system was established and systematically optimized in Klebsiella pneumoniae, enabling efficient and stable genome editing (75-100% efficiency). Using on this platform, by-product pathways were reduced through multi-gene deletions (frdA, poxB, adhE, ldhA, glpK, ptsG and dhaM) to enhance the yield of 1,3-PDO from glycerol. By heterologously expressing GPD1/GPP2 and optimizing promoter, a metabolic network for the "glucose-glycerol-1,3-PDO" pathways was reconstructed. Cometabolism studies indicated that low concentrations of xylose and arabinose as co-substrates enhanced conversion efficiency of glycerol, whereas glucose alleviated metabolic competition. Under cometabolism of glucose and glycerol, the engineered strain K. pneumoniae S2 ΔABEAKGM-1-2 produced 1003.7 mmol/L (76.4 g/L) of 1,3-PDO with a yield of 0.83 mol/mol glycerol, an overall molar yield of 0.78 mol/mol based on total substrate consumption, and a productivity of 27.9 mmol/L/h. When lignocellulosic hydrolysate was used as co-substrate, 981.1 mmol/L (74.7 g/L) of 1,3-PDO was produced with a yield of 0.77 mol/mol glycerol and a productivity of 27.3 mmol/L/h. This study achieved efficient redirection of carbon flux toward 1,3-PDO through systematic metabolic engineering, providing valuable strain resources and technical guidance for the sustainable and cost-effective biomanufacturing of bio-based 1,3-PDO.
Li Wang, Ming-Yang Zhao, Yuan-Ming Ye et al.· Bioresource Technology· 0 citations
Lacto-N-neotetraose (LNnT), a pivotal oligosaccharide in human milk, plays a significant role in modulating the intestinal microbiota of infants, and exhibits potential applications in antiviral therapy. Corynebacterium glutamicum is widely used in industrial fermentation, but it is incapable of synthesizing LNnT under natural conditions. Previously, we constructed a recombinant C. glutamicum strain CL014 for LNnT production, but the yield was limited. In this study, the key enzyme β-1,4-galactosyltransferase LgtB was engineered to improve LNnT biosynthesis in CL014. Fourteen mutant strains were constructed by replacing the critical residue Arg24 of LgtB to different amino acids and screened for the optimal LNnT production. The highest LNnT production was obtained in the mutant CL014-T. CL014-T could produce 1.40 g/L LNnT in shake flask cultivation, which is a 33.3% increase compared with the control strain CL014. Molecular docking simulations demonstrated that the mutation in CL014-T promotes the formation of additional hydrogen bonds in the flexible loop region, enhancing conformational flexibility and optimizing substrate interactions. Further mechanistic insights obtained through molecular dynamics simulations uncovered that the increased dynamic flexibility in two functional regions and the formation of more core hydrogen bonds collectively contribute to enhanced binding stability and improved product yield in the mutant. After 96 h feed-batch fermentation, CL014-T produced 2.29 g/L LNnT. This work demonstrates that C. glutamicum has the potential to be engineered as an efficient cell factory for human milk oligosaccharide production.
Ruyi Ma, Zihan Li, Chunyan Du et al.· Systems Microbiology and Bio...· 0 citations
Trehalose is a functional disaccharide widely used in the food, pharmaceutical, and cosmetic industries. It is industrially produced via a dual-enzyme process involving maltoligosaccharide trehalose synthase (MTSase) and maltoligosaccharide trehalose hydrolase (MTHase), with Escherichia coli (E. coli) serving as the expression host. Bacillus subtilis (B. subtilis) is an ideal host for industrial trehalose production due to its generally recognized as safe (GRAS) status and low phage susceptibility. However, engineered B. subtilis strains often exhibit slow growth, low heterologous protein expression, and high fermentation costs, thereby limiting their industrial application. To address these challenges, this study employed a synergistic strategy that combined chassis modification, expression element optimization, and knockout of substrate-competition pathways. First, a tryptophan-independent strain was constructed by reverting the trpC2 mutation to shorten the growth cycle. Next, knockout of flgD, yueB, and integration of E. coli-derived glutamate dehydrogenase (gdhA) significantly enhanced biomass accumulation. Expression of MTSase and MTHase was markedly improved through tandem strong promoters (PHpaII-P36) and ribosome-binding site (RBS) optimization (RBS1), achieving a 10.87-fold and 4.22-fold increase in enzyme activity, respectively. Finally, disruption of the amyE gene reduced non-specific substrate degradation. Using maltodextrin as substrate, the final trehalose conversion rate reached 76%. This study constructed B. subtilis chassis cells that highly express MTHase and MTSase respectively, laying a foundation for subsequent industrial trehalose production.
Jianghua Chen, Yu-Jue Wang, Qiang Wang et al.· Fermentation· 0 citations
O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.
Si-Min Huang, Xu-Yue He, Ruo-Nan Wang et al.· Journal of Biotechnology· 0 citations
1,4-Butanediamine is an important raw material for the synthesis of engineering plastics such as Polyamide 46 with excellent performance, which is widely used in automotive, electronics and machinery manufacturing industries. In this study, Escherichia coli BL21(DE3) was used as the starting strain to systematically modify the 1,4-butanediamine biosynthesis pathway using a modular strategy. 1,4-Butanediamine biosynthesis was re-divided into two modules: 1,4-butanediamine production module and α-ketoglutaric acid production module. By controlling the expression intensity of genes pykF, ppc, aceEF, gltA, icdA and gdhA, argD, argCB, argJ, ODC10, a recombinant producing 1,4-butanediamine strain PKT was obtained, achieving a yield of 862.82 mg/L of 1,4-butanediamine, which increased 8.62-fold compared to that of Escherichia coli BL21(DE3). Then, the fermentation medium of PKT strain was optimized. After 24 h of fermentation, the yield of 1,4-butanediamine reached 1843.60 mg/L, representing a 16.23-fold increase over the original strain. Furthermore, non-target metabolomics analysis was used to analyze the changes of metabolites during the efficient synthesis of 1,4-butanediamine by the recombinant strain PKT. The results showed that the total number of differential metabolites detected was 653. Differential metabolite pathway analysis showed that there were 9 differential metabolites in the 1,4-butanediamine synthesis pathway, with 5 involved in its synthesis pathway and 4 in its degradation branch. These differences in metabolites provide a theoretical basis for the modification of recombinant strains. The green production of 1,4- butanediamine by microbial cell factories through fermentation is the future direction. This research provides a theoretical guidance of building high yield 1,4-butanediamine engineering strains.
Xiangxiang Sun, Zongda Li, Yan-Ling Sun et al.· Journal of Genetic Engineeri...· 0 citations
Industrial enzymes are widely used in diverse applications, but low productivity limits their further widespread utilization. This research aimed to develop high-performance alkaline protease (AprE) expression strains of Bacillus licheniformis through element optimization and modular engineering. Firstly, the aprE gene expression cassette was systematically optimized through element engineering. To minimize host background interference, five large gene fragments were deleted from the genome of B. licheniformis DW2. This expression cassette and genome-reduced strain resulted in 5.77-, 4.84- and 1.31-fold increases in the activities of alkaline protease, nattokinase and chitinase, respectively. Crucially, metabolomics analysis then served as the pivotal discovery tool, revealing that high expression of AprE was constrained by insufficient precursor amino acids and excessive metabolic overflow. Subsequently, the amino acid biosynthesis, energy metabolism, overflow metabolism, and cell membrane/wall modules of the strain were successively modified. The final AprE expression host DM6E10 achieved a remarkable enzyme activity of 34,343 U/mL, with a maximum activity of 107,100 U/mL in a 5-L bioreactor. This study built an efficient cell factory for AprE production and provided insights for the optimization of other protein expression hosts.
Qing Zhang, Mengyuan Zhang, Zhihao Zhu et al.· Synthetic and Systems Biotec...· 0 citations
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