Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Phenotype-genotype mapping reveals the betaine-triggered L-arginine overproduction mechanism in Escherichia coli.

The production phenotype improvement of industrial microbes is extremely needed and challenging. Environmental factors optimization provides insightful ideas to trigger the superior production phenotype by activating potential genetic determiners. Here, phenotype-genotype mapping was used to dissect the betaine-triggered L-arginine overproduction mechanism and mine beneficial genes for further improving production phenotype. The comparative transcriptomic analysis revealed a novel role for betaine in modulating global gene transcription. Guided by this finding, 4 novel genes (cynX, cynT, pyrB, and rhaB) for L-arginine biosynthesis were identified via reverse engineering. Moreover, the rhaB deletion was demonstrated as a common metabolic engineering strategy to improve ATP pool in E. coli. By combinatorial genes manipulation, the L-arginine titer and yield increased by 17.9% and 28.9% in a 5-L bioreactor without betaine addition. This study revealed the molecular mechanism of gene transcription regulation by betaine and developed a superior L-arginine overproducer that does not require betaine.

Metabolic reprogramming and biosensor-assisted mutagenesis screening for high-level production of L-arginine in Escherichia coli.

L-arginine is a value-added amino acid with promising applications in the pharmaceutical and nutraceutical industries. Further unleashing the potential of microbial cell factories to make L-arginine production more competitive remains challenging due to the sophisticated intracellular interaction networks and the insufficient knowledge of global metabolic regulation. Here, we combined multilevel rational metabolic engineering with biosensor-assisted mutagenesis screening to exploit the L-arginine production potential of Escherichia coli. First, multiple metabolic pathways were systematically reprogrammed to redirect the metabolic flux into L-arginine synthesis, including the L-arginine biosynthesis, TCA cycle, and L-arginine export. Specifically, a toggle switch responding to special cellular physiological conditions was designed to dynamically control the expression of sucA and pull more carbon flux from the TCA cycle toward L-arginine biosynthesis. Subsequently, a biosensor-assisted high-throughput screening platform was designed and applied to further exploit the L-arginine production potential. The best-engineered ARG28 strain produced 132 g/L L-arginine in a 5-L bioreactor with a yield of 0.51 g/g glucose and productivity of 2.75 g/(L ⋅ h), which were the highest values reported so far. Through whole genome sequencing and reverse engineering, Frc frameshift mutant, PqiB A78P mutant, and RpoB P564T mutant were revealed for enhancing the L-arginine biosynthesis. Our study exhibited the power of coupling rational metabolic reprogramming and biosensor-assisted mutagenesis screening to unleash the cellular potential for value-added metabolite production.

Metabolic engineering of Escherichia coli for efficient osmotic stress-free production of compatible solute hydroxyectoine.

Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline.

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.

Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli.

Microbial production of l-tryptophan (l-trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate-limiting enzymes and blockage of competing pathways can effectively promote microbial production of l-trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of l-trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) pools in an l-trp producing strain of Escherichia coli for efficient l-trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the l-trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP-independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved l-trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP-pyruvate-oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L l-trp with an overall yield of 0.227 g/g glucose after 40 h fed-batch fermentation in 5-L bioreactor. This is the highest overall yield of l-trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient l-trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.

Identification and biochemical characterisation of a novel methionine aminopeptidase from the taiga tick Ixodes persulcatus.

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms and considered to be a valuable targets for the treatment of various diseases, including cancer, malaria, and bacterial infections. However, MetAPs have not been reported in hard ticks (family Ixodidae), and their bioinformatics characterisation in tick's genome sequences is limited. In this study, we cloned, identified, and characterised a novel MetAP from Ixodes persulcatus, a vector for pathogens causing Lyme borreliosis and tick-borne encephalitis. The sequence analysis showed that I. persulcatus MetAP was a type 1 enzyme carrying C-terminal motifs conserved in the M24A family of metallopeptidases. Protein-protein docking simulations using human MetAP revealed conservation of substrate and metal-binding residues in the catalytic site cleft of the novel enzyme, which was designated IpMetAP. Recombinant IpMetAP expressed in Escherichia coli revealed its significant enzymatic activity with the synthetic substrate H-Met-4-methyl-coumaryl-7-amide at pH 7.5 with Km of 0.014 mM, kcat of 0.25 s-1, and overall catalytic efficiency (kcat/Km) of 18.36 mM-1 s-1. The activity of IpMetAP was enhanced by the addition of divalent cations Mn2+ and Co2+ and significantly inhibited by EDTA and bestatin. Site-directed mutagenesis of conserved amino acids indicated that the substitution of metal-binding residues D226 and H288 completely abolished the IpMetAP enzymatic activity, whereas that of the other sites had only moderate effects on substrate hydrolysis. The catalytic properties of IpMetAP suggest that the enzyme behaves similar to other MetAPs and such characterization expands our knowledge of aminopeptidases and protein metabolism of tick.

High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation.

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3'-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.

Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli.

l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.

Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli.

Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3-4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.

Metabolic engineering of Escherichia coli for high-yield uridine production.

Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3 g/L uridine and 4.5 g/L uracil in shake flask culture for 32 h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8 g/L. As carbamyl phosphate, aspartate, and 5'-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5 g/L. After 64 h of fed-batch fermentation, the final engineered strain UR6 produced 70.3 g/L uridine with a yield and productivity of 0.259 g/g glucose and 1.1 g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin.

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening.

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5'-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

Synthesis and magnetic properties of manganite multiple heterostructure nanoribbons.

The fabrication and applications of two-dimensional complex oxide heterostructures have gained great attention. However, the achievement of these materials in one-dimensional form with multiple interfaces is still elusive. Here, we report the growth of manganite CaMn(3)O(6)/CaMn(2)O(4) heterostructure nanoribbons via the use of CaMnO(3) powders as the precursor for the molten-salt process. In contrast with the antiferromagnetism in CaMn(3)O(6) and CaMn(2)O(4) in the bulk, magnetization measurements indicate the coexistence of a ferromagnetic phase with a spin-glass-like component in CaMn(3)O(6)/CaMn(2)O(4) heterostructure nanoribbons. An asymmetric magnetization hysteresis loop observed in the applied magnetic field H≤ 3 T is attributed to the coupling between the antiferromagnetic phase and the ferromagnetic or spin-glass-like phase in CaMn(3)O(6)/CaMn(2)O(4) heterostructure nanoribbons.

[Characterization of pure and doped amorphous WO3 films by Raman spectroscopy].

Pure, TiO2-doped and TiO2/Ag-doped WO3 films were prepared by evaporation and electron beam evaporating. Raman spectroscopy and chronoamperometry were used to characterize the electrochromic properties of the samples. The correlation between the relative intensity of the Raman peaks, corresponding to the Raman sharp peak of the crystalline phase at 810 cm(-1) is negative, that is to say the higher the relative intensity of the Raman peaks, the smaller the coloration efficiency.

Automated method for optimization of electric field distributions and optical parameters in thin-film polarizers.

An efficient method based on the modified needle optimization technique is proposed to design high-power laser thin-film polarizers. In order to minimize the influence of the standing-wave electric field on the laser-induced damage threshold of the polarizers, a crucial optimization parameter, the maximum electric field intensity in the high-refractive-index layers, is included in the proposed merit function. The electric field distribution and optical performance obtained by the proposed method are studied. Improved electric field and identical optical characteristics are observed in comparison with those of the designs obtained by optimizing the traditional merit function without a standing-wave electric field term and by the analytical synthesis method.