Efficient Fermentative Production of d-Alanine and Other d-Amino Acids by Metabolically Engineered Corynebacterium glutamicum.

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.

Zeolitic Imidazole Framework (ZIF)-Sponge Composite for Highly Efficient U(VI) Elimination.

Herein, a zeolitic imidazole framework (ZIF-67) composite was prepared by a rapid, simple and inexpensive situ hybridization technique applying polyurethane sponge (PU) as support, which was designated as ZIF-67-PU. The ZIF-67 nanoparticle was successfully supported on the surface of sponge skeletons mainly through electrostatic attraction as well as probable π-π stacking interactions with PAM modification of the sponge. The resultant ZIF-67-PU exhibited a remarkably enhanced U(VI) elimination capacity of 150.86 mg∙g-1 on the basis of the Langmuir isotherm model, in comparison to pristine sponge. Additionally, the mechanism for U(VI) elimination was mainly achieved through the complex reaction between C-N(H)/-OH groups in ZIF-67 and U(VI), based on XPS investigations. ZIF-67-PU represents a simple, feasible and low-cost disposal option for preparing ZIF-coated sponges of any shape that can enhance the U(VI) elimination capacity. Furthermore, this approach can be widely applied to the preparation of various kinds of MOF-sponges through this situ hybridization technique.

Discovery and biochemical characterization of two hexokinases from Crassostrea gigas.

Hexokinases (HKs) play a vital role in glucose metabolism, which controls the first committed step catalyzing the production of glucose-6-phosphate from glucose. Two HKs (CGIHK1 and CGIHK2) from the Pacific oyster Crassostrea giga were cloned and characterized. CGIHK1 and CGIHK2 were recombinantly expressed in Escherichia coli and successfully purified by the Ni-NTA column. The optimum pH of the two enzymes was pH 8.0 and 8.5, respectively. The optimum temperature of the two enzymes was 42 °C and 50 °C, respectively. Both enzymes showed a clear requirement for divalent magnesium and were strongly inhibited by SDS. CGIHK1 exhibited highly strict substrate specificity to glucose, while CGIHK2 could also catalyze other 11 monosaccharide substrates. This is the first report on the in vitro biosynthesis of glucose-6-phosphate by the hexokinases from Crassostrea gigas. The facile expression and purification procedures combined with different substrate specificities make CGIHK1 and CGIHK2 candidates for the biosynthesis of glucose-6-phosphate and other sugar-phosphates.

Improve L-isoleucine production in Corynebacterium glutamicum WM001 by destructing the biosynthesis of trehalose dicorynomycolate.

Trehalose dicorynomycolates are structurally important constituents of the cell envelope in Corynebacterium glutamicum. The genes treS, treY, otsA, mytA and mytB are necessary for the biosynthesis of trehalose dicorynomycolates. In this study, the effect of biosynthesis of trehalose dicorynomycolates on L-isoleucine production in C. glutamicum has been investigated by deleting the genes treS, treY, otsA, mytA, and mytB in the L-isoleucine producing C. glutamicum WM001. L-isoleucine production was slightly improved in the mutants ΔtreY, ΔotsA, and ΔtreYA, and not improved in the single deletion mutant ΔtreS , but significantly improved in the triple deletion mutant ΔtreSYA. Deletion of mytA or mytB in ΔtreSYA could further improve L-isoleucine production. However, deletion of both mytA and mytB in ΔtreSYA significantly decreased L-isoleucine production. The final L-isoleucine producing C. glutamicum WL001 was constructed by deletion of treS, treY, otsA, and mytB, insertion of lrp, and replacement of the native promoter of ilvA with the L-isoleucine sensitive promoter PbrnFE7. WL001 grew worse than the control WM001, but produced 36.1% more L-isoleucine after 72 h shake flask cultivation than WM001.

The role of trehalose biosynthesis on mycolate composition and L-glutamate production in Corynebacterium glutamicum.

Corynebacterium glutamicum has been widely utilized for the industrial production of various amino acids. Trehalose is a prerequisite for the biosynthesis of mycolates which are structurally important constituents of the cell envelope in C. glutamicum. In this study, C. glutamicum mutant ΔSYA, which is unable to synthesize trehalose was constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis. In the fermentation medium, ΔSYA grew as well as the control C. glutamicum ATCC13869, synthesized similar levels of glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids to ATCC13869, but produced 12.5 times more L-glutamate than ATCC13869. Transcriptional levels of the genes relevant to L-arginine biosynthesis, encoding ODHC and relevant to the biosynthesis of sulfur-containing amino acids were down-regulated in ΔSYA. In minimal medium with different concentrations of glucose, ΔSYA grew worse than ATCC13869 but excreted more L-glutamate. When grown in minimal medium, phospholipids are the major lipid in ΔSYA, while glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids are the major lipid in ATCC13869. The transcriptional levels of mscCG in ΔSYA was significantly up-regulated when grown in minimal medium.

Antioxidant Interactions between S-allyl-L-cysteine and Polyphenols Using Interaction Index and Isobolographic Analysis.

This work aims to study the antioxidant interactions between S-allyl-L-cysteine (SAC) and six natural polyphenols (quercetin, caffeic acid, sinapic acid, catechin, ferulic acid, and 3,4-dihydroxybenzoic acid) through the measurement of free-radical-scavenging activity of 1,1-diphenyl- 2-picryl-hydrazyl (DPPH), the radical-cation-scavenging activity of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and reducing power. Among the six natural polyphenols, caffeic acid showed the strongest synergistic effect with SAC according to DPPH and reducing power assays. Further investigations based on the results of interaction index and isobologram analysis showed that the antioxidant activity (DPPH, ABTS, and reducing power) of the combination of caffeic acid with SAC presented an increase with the raising of their individual concentrations in their mixture and along with a dose-response manner. The best synergistic effect between caffeic acid and SAC based on DPPH, ABTS, and reducing power assays were observed at the ratio of 1:20, 1:35, and 1:70, respectively. The excellent synergic antioxidant activity of the combination of caffeic acid with SAC in our study suggests SAC has a more broad and effective application prospects in food field.

Physiological Responses of Ribosomal Protein S12 K43 Mutants of Corynebacterium glutamicum.

Bacterial resistance to streptomycin is often acquired as a consequence of mutations in rpsL, the gene encoding ribosomal protein S12. Corynebacterium glutamicum is a non-pathogenic Gram-positive soil bacterium that has been widely used in industry. In a previous study, we screened several streptomycin-resistant rpsL K43 mutants of C. glutamicum, and surprisingly found that two of them also confer chloramphenicol and/or kanamycin resistance. In order to understand whether or not a single mutation of rpsLK43 could confer resistance to multiple antibiotics, in this study we attempted to construct saturation mutagenesis of rpsL K43 by rational genetic manipulation. Despite many efforts had been made, only nine mutants were successfully constructed. They were indeed resistant to streptomycin, but not to other antibiotics. This suggested that other mutations should be acquired, contributing to multiple antibiotics in the screened strains. The growth and enhanced green fluorescent protein (eGFP) expression of these nine mutants were then investigated. The results showed that they grew differently in CGXII minimal medium, but not in BHI medium. When cultured in the absence of streptomycin, the expression of eGFP was positively proportional to the growth, approximately, while in the presence of streptomycin, the expression of eGFP was proportional to the ability of streptomycin resistance.

Optimization of the UDP-Xyl biocatalytic synthesis from Crassostrea gigas by orthogonal design method.

UDP-Xyl, a nucleotide sugar involved in the biosynthesis of various glycoconjugates, is difficult to obtain and quite expensive. Biocatalysis using a one-pot multi-enzyme cascade is one of the most valuable biotransformation processes widely used in the industry. Herein, two enzymes, UDP-glucose (UDP-Glc) dehydrogenase (CGIUGD) and UDP-Xyl synthase (CGIUXS) from the Pacific oyster Crassostrea gigas, which are coupled together for the biotransformation of UDP-Xyl, were characterized. The optimum pH was determined to be pH 9.0 for CGIUGD and pH 7.5 for CGIUXS. Both enzymes showed the highest activity at 37 °C. Neither enzyme is metal ion-dependent. On this basis, a single factor and orthogonal test were applied to optimize the condition of biotransformation of UDP-Xyl from UDP-Glc. Orthogonal design L9 (33) was conducted to optimize processing variables of enzyme amount, pH, and temperature. The conversion of UDP-Xyl was selected as an analysis indicator. Optimum variables were the ratio of CGIUGD to CGIUXS of 2:5, enzymatic pH of 8.0, and temperature of 37 °C, which is confirmed by three repeated validation experiments. The UDP-Xyl conversion was 69.921% in a 1 mL reaction mixture by optimized condition for 1 h. This is the first report for the biosynthesis of UDP-Xyl from oyster enzymes.

High-level production of l-homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering.

l-Homoserine is a valuable non-proteinogenic amino acid used in the synthesis of various important compounds. Microbial fermentation has potential value for producing l-homoserine on a large scale, but suffers from a low yield and the need for expensive additives. In this study, a non-induced, non-auxotrophic, plasmid-free Escherichia coli chassis for the high-efficiency production of l-homoserine was constructed. Initially, the l-homoserine degradation pathway was dynamically attenuated. Subsequently, systems metabolic engineering strategies were employed, including reinforcing the synthetic flux, improving NADPH generation, and elevating l-homoserine efflux. The constructed strain HOM-14, produced 60.1 g/L l-homoserine without additional supplements or inducers, which achieved the highest fermentative production efficiency of l-homoserine till date. Moreover, common byproducts, such as acetate, did not accumulate. The strategies presented here can be applied in the further engineering of chassis for the scale-up production of l-homoserine and derivatives.

Sequence and phylogenetic analysis of the mitochondrial genome for the Poyang Lake, Misgurnus anguillicaudatus (natural diploid loach).

Misgurnus anguillicaudatus is a highly valued, aquaculture-relevant food fish in East Asian countries. In this study, the complete mitochondrial genome of Poyang loach was obtained by PCR. The genome is 16,646 bp in length, including 2 ribosomal RNA genes. 13 protein-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of the species were similar to most reported from other vertebrates. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Poyang M. anguillicaudatus, and the other loaches had their own branches.

Liposomal Spherical Nucleic Acid Scaffolded Site-Selective Hybridization of Nanoparticles for Visual Detection of MicroRNAs.

In this study, the advanced liposomal spherical nucleic acid (L-SNA) is exploited for the first time to establish a spherical, three-dimensional biosensing platform by hybridizing with a set of nanoparticles. By hydrophilic and hydrophobic interactions as well as programmable base-pairing, red-emission quantum dots (QDs), green-emission QDs, and gold nanoparticles (AuNPs) are encapsulated into the internal aqueous core, the intermediate lipid bilayer, and the outer SNA shell, respectively, producing an L-SNA-nanoparticle hybrid. As a result of the site-selective encapsulation, the hybrid constitutes a liposomal fluorescent "core-resonance energy transfer" system surrounded by a SNA shell, as is imaged at the single-particle resolution by confocal microscopy. With the outer SNA shell as three-dimensional substrate for duplex-specific nuclease target recycling reaction, the hybrid is capable of amplified detection of microRNAs, featuring one target to many AuNP-manipulated, dual-emission QD-based ratiometric fluorescence. More importantly, the ratiometric fluorescence facilitates the hybrid to visualize microRNAs with remarkably high resolution, which is exemplified by traffic light-type transition in fluorescence color for diagnosing circulating microRNAs in clinical serum samples. Substantially, the controllable hybridization with functional nanoparticles opens an avenue for the exciting biomedical applications of liposomal spherical nucleic acids.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR-based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid-mediated, the editing efficiency of which is low due to high false-positive rates of sacB counter selection, and the requirement for tedious two-round selection and verification of rare double-cross-over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two-step verification strategy can be simplified to just one-step verification of the final edited strain. As proof of concept, a 2.5-kb DNA fragment comprising aroGfbr pheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l-1  l-tyrosine in shake-flask fermentation. This updated suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes.

Effects of drying pretreatment and particle size adjustment on the composting process of discarded flue-cured tobacco leaves.

The main characteristic of discarded flue-cured tobacco leaves is their high nicotine content. Aerobic composting is an effective method to decrease the nicotine level in tobacco leaves and stabilize tobacco wastes. However, high levels of nicotine in discarded flue-cured tobacco leaves complicate tobacco waste composting. This work proposes a drying pretreatment process to reduce the nicotine content in discarded flue-cured tobacco leaves and thus enhance its carbon-to-nitrogen ratio to a suitable level for composting. The effect of another pretreatment method, particle size adjustment, on composting efficiency was also tested in this work. The results indicated that the air-dried (nicotine content: 1.35%) and relatively long discarded flue-cured tobacco leaves (25 mm) had a higher composting efficiency than damp (nicotine content: 1.57%) and short discarded flue-cured tobacco leaves (15 mm). When dry/25 mm discarded flue-cured tobacco leaves mixed with tobacco stems in an 8:2 ratio was composted at a temperature above 55 °C for 9 days, the nicotine content dropped from 1.29% to 0.28%. Since the discarded flue-cured tobacco leaves was successfully composted to a fertile and harmless material, the germination index values increased to 85.2%. The drying pretreatment and particle size adjustment offered ideal physical and chemical conditions to support microbial growth and bioactivity during the composting process, resulting in efficient conversion of discarded flue-cured tobacco leaves into a high quality and mature compost.

Optimization of ultrasound-assisted extraction of indigo and indirubin from Isatis indigotica Fort. and their antioxidant capacities.

An effective method for the ultrasound-assisted extraction of indigo and indirubin from Isatis indigotica Fort. was established and their antioxidant activities were investigated. Response surface methodology based on a three-level, four-factor Box-Behnken design was used to optimize the extraction conditions. Analysis of variance showed that the quadratic model was significant for the extraction of indigo and indirubin (112.72% ± 1.65% and 116.42% ± 1.27%, respectively) under the optimal conditions (methanol concentration, 80%; extraction time, 25 min; ratio of solid to liquid, 1:34 g/mL; and extraction temperature, 41 °C) and was in good agreement with the predicted value. Moreover, evaluation of the antioxidant activities suggested that indigo and indirubin presented better scavenging effects on 1,1-diphenyl-2-picrylhydrazyl free radical and superoxide radical than the extract and the extract revealed certain antioxidant activities in hydroxyl radical scavenging and reducing power, and indigo and indirubin could be used as natural antioxidants in the food or medicine industry.