Efficient Biosynthesis of Difucosyllactose via De Novo GDP-l-Fucose Pathway in Metabolically Engineered Escherichia coli.

Difucosyllactose (DFL) is an important component of human milk oligosaccharides (HMOs) and has significant benefits for the growth and development of infants. So far, a few microbial cell factories have been constructed for the production of DFL, which still have problems of low production and high cost. Herein, a high-level de novo pathway DFL-producing strain was constructed by multistep optimization strategies in Escherichia coli BL21star(DE3). We first efficiently synthesized the intermediate 2'-fucosyllactose (2'-FL) in E. coli BL21star(DE3) by the advisable stepwise strategy. The truncated α-1,3/4-fucosyltransferase (Hp3/4FT) was then introduced into the engineered strain to achieve de novo biosynthesis of DFL. ATP-dependent protease (Lon) and GDP-mannose hydrolase (NudK) were deleted, and mannose-6-phosphate isomerase (ManA) was overexpressed to improve GDP-l-fucose accumulation. The regulator RcsA was overexpressed to fine-tune the expression level of pathway genes, thereby increasing the synthesis of DFL. The final strain produced 6.19 g/L of DFL in the shake flask and 33.45 g/L of DFL in the 5 L fermenter, which were the highest reported titers so far. This study provides a more economical, sustainable, and effective strategy to produce the fucosylated human milk oligosaccharides (HMOs).

Construction of an engineered Escherichia coli for effective synthesis of 2'-fucosyllactose via the salvage pathway.

2'-Fucosyllactose (2'-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2'-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2'-FL. The 2'-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2'-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2'-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2'-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2'-FL or other HMOs.

Response mechanism of Vibrio parahaemolyticus at high pressure revealed by transcriptomic analysis.

Vibrio parahaemolyticus is a common pathogen in aquatic products, such as shellfishes. Laboratory-based simulated studies demonstrated that V. parahaemolyticus can tolerate high hydrostatic pressure (HHP) up to 20 MPa. However, the molecular mechanisms of high-pressure adaptation remain unclear. Herein, we analyzed the physiological changes and transcriptomic responses of V. parahaemolyticus ATCC 17,802 under HHP conditions to determine the possible survival mechanisms. Under HHP conditions, the morphology of V. parahaemolyticus was notably changed exhibiting the coccoid microbial cells. The transcriptome analysis revealed that there were 795 differentially expressed genes (DEGs) under the 20 MPa condition, including 406 upregulated DEGs and 389 downregulated DEGs. Most of the downregulated DEGs encoded proteins related to energy metabolism, such as citrate synthase (gltA), pyruvate kinase (pyk), and glyceraldehyde-3-phosphate dehydrogenase (gapA). Many of the upregulated DEGs encoded proteins related to adhesion and virulence factors, such as RNA polymerase σ factor (rpoE), L-threonine 3-dehydrogenase, and bacterial nucleotide signal c-di-GMP (WU75_RS02745 and WU75_RS07185). In our proposed mechanism model, V. parahaemolyticus responds to HHP stress through RNA polymerase σ factor RpoE. These findings indicate that V. parahaemolyticus cells may adopt a complex adaptation strategy to cope with HHP stress. KEY POINTS: •The transcriptomic response of Vibrio parahaemolyticus under HHP conditions was studied for the first time. •V. parahaemolyticus may adopt a complex adaptation strategy to cope with HHP stress. •ToxRS and RpoE played an important role in sensing and responding the HHP signal.

Partially degraded chitosan-based flocculation to achieve effective deodorization of oyster (Crassostrea gigas) hydrolysates.

Aquatic protein hydrolysates are usually associated with unpleasant odors and high fat content, which seriously restricts their industrial utilization. In this study, chitosans with different molecular weights produced by hydrogen peroxide degradation were applied to establish a flocculation method, using for the deodorization and defatting of oyster (Crassostrea gigas) hydrolysates. GC-MS analysis showed that the method markedly decreased the content of the fishy odor constituents. Up to 92 % fat and part of the heavy metals were effectively removed. Protein recovery percentage and solid recovery percentage were 83.43 ± 0.35 % and 76.36 ± 0.52 %, respectively, at the optimum dose (150 mg/L) of chitosan (83 % of deacetylation degree, 77 kDa). Thus, chitosan flocculation-coupled centrifugation (5000g, 1 min) can effectively solve the current drawbacks of engineering disc centrifuges and can be industrially used for defatting and deodorization during aquatic food processing.