Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.
Escherichia coli/genetics* ; NAD ; Succinic Acid ; Acetic Acid ; Adenosine Triphosphate

Salicylate 2-O-β-d-glucoside (SAG) is a derivative of salicylate in plants. Recent reports showed that SAG could be considered as a potential anti-inflammatory substance due to its anti-inflammatory and analgesic effects, and less irritation compared with salicylic acid and aspirin. The biological method uses renewable resources to produce salicylic acid compounds, which is more environmentally friendly than traditional industry methods. In this study, Escherichia coli Tyr002 was used as the starting strain, and a salicylic acid producing strain of E. coli was constructed by introducing the isochorismate pyruvate lyase gene pchB from Pseudomonas aeruginosa. By regulating the expression of the key genes in the downstream aromatic amino acid metabolic pathways, the titer of salicylic acid reached 1.05 g/L in shake flask fermentation. Subsequently, an exogenous salicylic acid glycosyltransferase was introduced into the salicylic acid producing strain to glycosylate the salicylic acid. The newly engineered strain produced 5.7 g/L SAG in shake flask fermentation. In the subsequent batch fed fermentation in a 5 L fermentation tank, the titer of SAG reached 36.5 g/L, which is the highest titer reported to date. This work provides a new route for biosynthesis of salicylate and its derivatives.
Escherichia coli/genetics* ; Glucosides ; Metabolic Engineering ; Salicylic Acid ; Pyruvic Acid

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.
Humans ; Methionine ; Methylenetetrahydrofolate Reductase (NADPH2) ; Carbon ; Cysteine ; Escherichia coli/genetics* ; Hydroxymethyl and Formyl Transferases ; Carrier Proteins ; Escherichia coli Proteins

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.
Thermomonospora ; Actinomycetales ; Escherichia coli/genetics* ; Polyhydroxyalkanoates

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10^F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO^{10F138L/D218G}-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD₆₀₀ of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD₆₀₀ was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.
Escherichia coli/genetics* ; Biological Products ; Bioreactors ; Fermentation

Soluble cello-oligosaccharide with 2-6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2-6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD₆₀₀) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.
Humans ; Oligosaccharides ; Biomass ; Escherichia coli/genetics* ; Gastrointestinal Microbiome ; Glucose

Pullulanase is a starch debranching enzyme, which is difficult in secretory expression due to its large molecular weight. Vibrio natriegens is a novel expression host with excellent efficiency in protein synthesis. In this study, we achieved secretory expression of the full-length pullulanase PulA and its truncated mutant PulN2 using V. natriegens VnDX strain. Subsequently, we investigated the effects of signal peptide, fermentation temperature, inducer concentration, glycine concentration and fermentation time on the secretory expression. Moreover, the extracellular enzyme activities of the two pullulanases produced in V. natriegens VnDX and E. coli BL21(DE3) were compared. The highest extracellular enzyme activity of PulA and PulN2 in V. natriegens VnDX were 61.6 U/mL and 64.3 U/mL, which were 110% and 62% that of those in E. coli BL21(DE3), respectively. The results indicated that V. natriegens VnDX can be used for secretory expression of the full-length PulA with large molecular weight, which may provide a reference for the secretory expression of other large molecular weight proteins in V. natriegens VnDX.
Escherichia coli/genetics* ; Fermentation ; Vibrio/genetics*

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×10⁴ CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×10⁶ CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.
Electroporation ; Electroporation Therapies ; Bacillaceae ; Cell Membrane ; Escherichia coli/genetics*

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg²⁺, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.
Child ; Humans ; Child, Preschool ; N-Acetylneuraminic Acid ; Escherichia coli/genetics* ; Lactose ; Fermentation ; Saccharomyces cerevisiae ; Oligosaccharides ; Glucosamine

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay