Search

Saline-alkali stress seriously affects the yield and quality of crops, threatening food security and ecological security. Improving saline-alkali land and increasing effective cultivated land are conducive to sustainable agricultural development. Trehalose, a nonreducing disaccharide, is closely related to plant growth and development and stress response. Trehalose 6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) are key enzymes catalyzing trehalose biosynthesis. To elucidate the effects of long-term saline-alkali stress on trehalose synthesis and metabolism, we conducted an integrated transcriptome and metabolome analysis. As a result, 13 TPS and 11 TPP genes were identified in quinoa (Chenopodium quinoa Willd.) and were named CqTPS1-13 and CqTPP1-11 according to the order of their Gene IDs. Through phylogenetic analysis, the CqTPS family is divided into two classes, and the CqTPP family is divided into three classes. Analyses of physicochemical properties, gene structures, conservative domains and motifs in the proteins, and cis-regulatory elements, as well as evolutionary relationships, indicate that the TPS and TPP family characteristics are highly conserved in quinoa. Transcriptome and metabolome analyses of the sucrose and starch metabolism pathway in leaves undergoing saline-alkali stress indicate that CqTPP and Class II CqTPS genes are involved in the stress response. Moreover, the accumulation of some metabolites and the expression of many regulatory genes in the trehalose biosynthesis pathway changed significantly, suggesting the metabolic process is important for the saline-alkali stress response in quinoa.

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the -10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.

Summary Electrocatalytic CO2 reduction reaction (CO2RR) has attained great attention as it can transform the greenhouse gas CO2 to value-added chemicals, such as carbon monoxide, formic acid, ethylene, ethanol, and n-propanol. However, the electrocatalytic performance of materials, especially the metal materials, toward the CO2RR is still limited due to the complex reaction processes and broad product distribution. Very recently, surface modification of metal materials has emerged as an effective way to boost their electrocatalytic CO2RR performance. Herein, recent advances in the surface modification of metal materials, which includes surface molecular functionalization, surface elemental doping, and surface plasma treatment, for CO2RR are presented. The surface modification-induced variations of surface states and electrocatalytic performances of metal materials, together with the underlying mechanisms, are highlighted. It is believed that the development of surface modification could promote the rational design and preparation of advanced CO2RR electrocatalysts toward practical applications.

Synonymous mutation of the N-terminal coding sequence (NCS) has been used to regulate gene expression. We here developed a statistical model to predict the effect of the NCSs on protein expression in Bacillus subtilis WB600. First, a synonymous mutation was performed within the first 10 residues of a superfolder green fluorescent protein to generate a library of 172 NCS synonymous mutants with different expression levels. A prediction model was then developed, which adopted G/C frequency at the third position of each codon and minimum free energy of mRNA as the independent variables, using multiple regression analysis between the 11 sequence parameters of the NCS and their fluorescence intensities. By designing the NCS of the 10 signal peptides de novo according to the model, the extracellular yield of B. subtilis pullulanase fused to each signal peptide was up-regulated by up to 515% or down-regulated by at most 79%. This work provided a candidate tool for fine-tuning gene expression or enzyme production in B. subtilis.

Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Fusion with self-assembling amphipathic peptides (SAPs) could improve enzyme stability and activity in addition to their effects on protein expression. In this study, we proposed a metabolic regulation strategy based on an iterative SAP fusion with key enzymes in pathway. First, an SAP, S1nv10 (ANANARARANANARAR), was separately fused to the N-terminus of metabolite enzymes. To achieve the diversity in the enzyme activities, a library of the fused SAPs varied in hydrophobicity, length and net charge were generated by a multi-primer PCR procedure and the DATEL-assembly method. After simultaneous modification of three enzymes (CrtE, CrtY, and CrtI) using the SAP library, the Escherichia coli strain with 3.91-fold increase in β-carotene production was isolated. As indicated by western-blot and catalytic property analysis, SAP fusion induced the increases in both enzyme expression (CrtY and CrtE) and enzymatic activities (CrtY). The effectiveness of the strategy was further confirmed by the metabolic regulation of the eriodictyol and (2S)-naringenin, the yields of which were increased by 111.3% and 186.3% in E. coli, respectively. These results indicated that fusion with SAPs could effectively regulate metabolic pathway through optimization of the protein expression and catalytic properties of the key enzymes.

Over the past few decades, gold-based materials have drawn extensive attention due to their intriguing physical/chemical properties and excellent performance in a broad range of applications. In particular, the remarkable technical progress in synthesis and characterization is promoting the rapid development of gold-based nanoalloys, which are of great significance in catalysis. In this review, we provide a brief overview of the recently reported gold-based nanoalloys, focusing on their general synthetic methods and potential catalytic applications. In particular, the general relationships between material structures/compositions and catalytic performances are discussed in representative chemical reactions, such as the carbon dioxide reduction reaction (CO2RR), oxygen reduction reaction (ORR), hydrogen evolution reaction (HER), oxygen evolution reaction (OER), small organic molecule oxidation reaction, carbon monoxide oxidation reaction, and typical heterogeneous catalytic reactions in chemical engineering. Furthermore, critical challenges and potential opportunities facing gold-based nanoalloys are proposed to provide possible inspiration for future material design towards high performance applications.

Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8 mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR NK cell efficacy. It has been reported that mesothelin (MSLN) may be an ideal immunotherapy target for gastric cancer. However, the feasibility of using anti-MSLN CAR NK cells to treat gastric cancer remains to be studied. Methods: MSLN expression in primary human gastric cancer, normal tissues and cell lines were detected. MSLN and CD19 targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed, purified and verified. N87, MKN-28, AGS and Huh-7 cells expressing the GFP and luciferase genes were transduced. Cell- and patient-derived xenograft (PDX) were established via NSG mice. The ability of MSLN-CAR NK cells to kill MSLN-positive gastric cancer cells were evaluated in vitro and in vivo. Results: MSLN-CAR NK cells can specifically kill MSLN-positive gastric cancer cells (N87, MKN-28 and AGS), rather than MSLN negative cell (Huh-7), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretions were secreted in MSLN-CAR NK cells cocultured with N87, MKN-28 and AGS. Furthermore, MSLN-CAR NK cells can effectively eliminate gastric cancer cells in both subcutaneous and intraperitoneal tumor models. They could also significantly prolong the survival of intraperitoneally tumor-bearing mice. More importantly, the potent antitumor effect and considerable NK cell infiltration were observed in the patient-derived xenograft treated with MSLN-CAR NK cells, which further warranted the therapeutic effects of MSLN-CAR NK cells to treat gastric cancer. Conclusion: These results demonstrate that MSLN-CAR NK cells possess strong antitumor activity and represent a promising therapeutic approach to gastric cancer.

Patatin is a useful plant protein with excellent gelation properties that could be used as a gelling agent in the food industry. However, the commercial production of patatin is limited because the traditional extraction methods are inefficient and time consuming. Production of patatin with gelation properties by microorganisms is a promising alternative route. In this study, 1424.5 mg/L patatin storage protein with great gelation properties could be obtained in a 5-L bioreactor after optimization of the signal peptide, the promoter, and the fed-batch process when a Pichia pastoris GS115, but not Escherichia coli, expression system was used. Compared with commercial potato-extracted patatins, P. pastoris-derived patatins showed better gelation properties, such as a lower gel-forming concentration and gelation temperature. In addition, the gel strength of P. pastoris-derived patatins was comparable with that of potato-extracted patatins. These results suggested that P. pastoris-derived patatins have the potential to replace current potato-derived ones, which are now widely used in plant-based meat products.

(2

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from

Because of the distinct electronic structures and stable chemical properties, rhodium materials are one of the most important noble metal catalysts for various kinds of industrial chemical processe...

The mechanistic understanding of hydrogenolysis of asymmetric Caryl–O–Calkyl bonds is key to the rational design of high-performance catalysts for bio-oil upgrading. Herein, we theoretically explor...

The compound (2S)-eriodictyol is an important flavonoid that can be derived from (2S)-naringenin through flavonoid 3'-hydroxylase (F3'H) catalyzation. F3'H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. However, P450s have limited applications in industrial scale biosynthesis, owing to their low activity. Here, an efficient SmF3'H and a matched SmCPR were identified from Silybum marianum. To improve the efficiency of SmF3'H, we established a high-throughput detection method for (2S)-eriodictyol, in which the promoter combination of SmF3'H and SmCPR were optimized in Saccharomyces cerevisiae. The results revealed that SmF3'H/SmCPR should be expressed by using promoters with similar and strong expression levels. Furthermore, directed evolution was applied to further improve the efficiency of SmF3'H/SmCPR. With the optimized promoter and mutated combinations SmF3'HD285N/SmCPRI453V, the (2S)-eriodictyol titer was improved to 3.3 g/L, the highest titer in currently available reports. These results indicated that S. cerevisiae is an ideal platform for functional expression of flavonoid related P450 enzymes.

Dihydromyricetin (DHM) is a traditional plant-extracted flavonoid with some health benefits. This study aimed to metabolically engineer the strains for DHM bioproduction. Two strains of BK-11 and BQ-21 were integrated with flavonoid 3-hydroxylase (F3H) or both F3H and flavonoid 3'-hydroxylase (F3'H). The resulting strains have expressed the enzymes of GmCPR and SlF3'5'H, and then, the promoters of INO1p and TDH1p were used to enhance further the DHM production from naringenin in Saccharomyces cerevisiae. Through multiple-copy integration, 709.6 mg/L DHM was obtained by adding 2.5 g/L naringenin in a 5 L bioreactor, implying that the synergistic effect between F3'H and flavonoid 3'5'-hydroxylase is likely to promote the DHM production. An yield of 246.4 mg/L DHM was obtained from glucose by deleting genes for branch pathways and integrating PhCHS, MsCHI, Pc4CL, and FjTAL. To our knowledge, this is the highest production reported for the de novo biosynthesis of DHM.

Flavonoids have diverse biological functions in human health. All flavonoids contain a common 2-phenyl chromone structure (C6-C3-C6) as a scaffold. Hence, in using such a scaffold, plenty of highvalue-added flavonoids can be synthesized by chemical or biological catalyzation approaches. (2S)-Naringenin is one of the most commonly used flavonoid scaffolds. However, biosynthesizing (2S)-naringenin has been restricted not only by low production but also by the expensive precursors and inducers that are used. Herein, we established an induction-free system to de novo biosynthesize (2S)-naringenin in Escherichia coli. The tyrosine synthesis pathway was enhanced by overexpressing feedback inhibition-resistant genes (aroGfbr and tyrAfbr) and knocking out a repressor gene (tyrR). After optimizing the fermentation medium and conditions, we found that glycerol, glucose, fatty acids, potassium acetate, temperature, and initial pH are important for producing (2S)-naringenin. Using the optimum fermentation medium and conditions, our best strain, Nar-17LM1, could produce 588 mg/l (2S)-naringenin from glucose in a 5-L bioreactor, the highest titer reported to date in E. coli.

Lipoxygenases (LOXs) are a family of non-heme iron oxidoreductases, which catalyze the addition of oxygen into polyunsaturated fatty acids. They have applications in the food and medical industries. In most studies, the soluble expression of LOXs in microbes requires low temperature ( 20 °C), which increases the cost and fermentation time. Achievement of soluble expression in elevated temperatures ( 30 °C) would shorten the production phase, leading to cost-efficient industrial applications. In this study, a combinatorial strategy was used to enhance the expression of soluble LOXs, comprising plasmid stability systems plus optimized carbon source used for auto-induction expression. Plasmid stability analysis suggested that both active partition systems and plasmid-dependent systems were essential for plasmid stability. Among them, the parBCA in it resulted in the enzyme activity increasing by a factor of 2 (498 ± 13 units per gram dry cell weight (U/g-DCW) after 6-h induction). Furthermore, the optimized carbon source, composed of glucose, lactose, and glycerol, could be used as an auto-induction expression medium and effectively improve the total and soluble expression of LOX, which resulted in the soluble expression of LOX increased by 7 times. Finally, the soluble expression of LOX was 11 times higher with a combinatorial strategy that included both optimized plasmid partition and auto-induction medium. Our work provides a broad, generalizable, and combinatorial strategy for the efficient production of heterologous proteins at elevated temperatures in the E. coli system. KEY POINTS : • Soluble expression of lipoxygenase at 30 °C or higher temperatures is industrially beneficial. • Strategies comprise plasmid partition and optimized auto-induction medium with glucose, lactose, and glycerol as carbon source. • Combinatorial strategy further improved LOX soluble expression at 30 °C and 37 °C.

Long-term exposure to fine particulate matter (PM2.5) is associated with kidney dysfunction. However, few studies have investigated acute effects of PM2.5 elemental constituents on renal function. We evaluated associations between personal PM2.5 and its elemental constituents and kidney function, assessed by an estimated glomerular filtration rate (eGFR) in Biomarkers of Air Pollutants Exposure in the Chinese aged 60-69 study. Seventy one older individuals were visited monthly between September 2018 and January 2019. Each participant wore a PM2.5 monitor for 72 h, responded to a questionnaire, and underwent a physical examination with blood sampling. Linear mixed-effect models were used to estimate associations between personal PM2.5 elemental constituents and eGFR. We found that significant changes in eGFR from -1.69% [95% confidence interval (CI): -3.34%, -0.01%] to -3.27% (95% CI: -5.04%, -1.47%) were associated with interquartile range (IQR) increases in individual PM2.5 exposures at various lag periods (7-12, 13-24, 0-24, 25-48, and 49-72 h). An IQR increase in 72 h moving averages of copper, manganese, and titanium in personal PM2.5 corresponded to -2.34% (95% CI: -3.67%, -0.99%) to -4.56% (95% CI: -7.04%, -2.00%) changes in eGFR. Personal PM2.5 and some of its elemental constituents are inversely associated with eGFR in older individuals.

Pyridoxal 5′-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA–PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET–TPL–pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21–TPLST–Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 μM.

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.

Integrin plays a prominent role in neurite outgrowth by transmitting both mechanical and chemical signals. Integrin expression is closely associated with Astragaloside IV (AS-IV), the main component extracted from Astragali radix, which has a positive effect on neural-protection. However, the relationship between AS-IV and neurite outgrowth has not been studied exhaustively to date. The present study investigated the underlying mechanism of AS-IV on neurite outgrowth. Longer neurites have been observed in SH-SY5Y cells or cortical neurons after AS-IV treatment. Furthermore, AS-IV not only increased the expression of integrin β but also activated it. The AS-IV-induced increased integrin activity was attributed to the integrin-activating protein talin. Application of the actin force probe showed that AS-IV led to an increase in intracellular microfilament force during neurite growth. Furthermore, in response to AS-IV, the microfilament force was regulated by talin and integrin activity during neurite growth. These results suggest that AS-IV has the ability to increase intracellular structural force and facilitate neurite elongation by integrin signaling, which highlights its therapeutic potential for neurite outgrowth.

Industrial production of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C, is mainly achieved by a two-step fermentation process carried out by Gluconobacter oxydans, Bacillus, and Ketogulonicigenium. One of the most promising innovations that could replace this complicated two-step fermentation process is the integration of the essential genes for synthesis of 2-KLG into G. oxydans and use of it as the producer. Therefore, determining the tolerance and response of G. oxydans to 2-KLG is a priority for improving the direct production of 2-KLG in this bacterium. In this study, a global view of the gene expression of G. oxydans WSH-003 in response to 2-KLG challenge was investigated by RNA sequencing. A total of 363 genes of G. oxydans that were differentially expressed in response to 2-KLG were uncovered. The results showed that 2-KLG could lead to oxidative stress, osmotic stress, and DNA damage in G. oxydans.

Cattleyak, which are interspecific hybrids between cattle and yak, display much higher growth performances than yak. However, F1 male cattleyak are infertile due to defective testicular development. Sirtuin 1 (SIRT1) is a histone deacetylase that is essential for various biological processes, while the roles of testicular SIRT1 in yak and cattleyak are still poorly understood. Here, we found that SIRT1 was localized in various kinds of yak testicular cells except elongated spermatids while it was deficient in cattleyak testis. Further studies indicated that cattleyak testis exhibited decreased histone acetylation levels on H3 and H4. One of SIRT1 co-factors, steroidogenic factor-1 (SF-1), was lost in cattleyak testis at protein level. Expressions of several SF-1 target genes responsible for Sertoli cell development and steroidogenesis, including STAR, CYP11A1, CYP26B1, FDX1 and HSD3B, decreased significantly in cattleyak testis. In addition, SIRT1-mediated P53 acetylation was not responsible for the cell apoptosis in cattleyak testis. Taken together, our results suggested the deficiency of SIRT1 in yak testis caused inactivation of SF-1 and the impairment of testicular development. This research provides theoretical bases for understanding the mechanism of cattleyak sterility and gives new insights in revealing the roles of SIRT1 in regulating yak testicular development.

Enzyme technologies are widely used in the food industry due to their advantages of high efficiency, specificity, and safety. Recently, “future foods” is emerging as a new research hotspot with healthier foods that are more nutritious, delicious, and sustainable; however, these foods still have problems with texture, nutrition, and flavor. Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects. In this review, we summarize enzyme technology applications in future food production, focusing on food texture, safety, and flavors. Furthermore, we discuss the prospects of enzyme-based technologies for future food production, including the modification of enzyme activities, the development of suitable food-grade hosts for enzyme production, and the optimization synergistic multi-enzyme systems.

The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA. Electronic supplementary material The online version of this article (10.1007/s10295-020-02294-4) contains supplementary material, which is available to authorized users.

There are numerous microorganisms in nature capable of synthesizing diverse useful compounds; however, these natural microorganisms are generally inefficient in the production of target products on an industrial scale, relative to either chemical synthesis or extraction methods. To achieve industrial production of useful compounds, these natural microorganisms must undergo a certain degree of mutation or effective fine-tuning strategies. This review describes how to achieve an ideal metabolic fine-tuned process, including static control strategies and dynamic control strategies. The static control strategies mainly focus on various matabolic engineering strategies, including protein engineering, upregulation/downregulation, and combinatrorial control of these metabolic engineering strategies, to enhance the flexibility of their application in fine-tuned metabolic metworks. Then, we focus on the dynamic control strategies for fine-tuned metabolic metworks. The design principles derived would guide us to construct microbial cell factories for various useful compounds.

Pathway optimization plays an important role in fine-tuning metabolic pathways. In most conditions, more than three genes are involved in the biosynthesis pathway of a specific target product. To improve the titer of products, rational regulation of a group of genes by a series of promoters with different strengths is essential. On the basis of a series of RNA-Seq data, a set of 66 native promoters was chosen to fine-tune gene expression in Saccharomyces cerevisiae. Promoter strength was characterized by measuring the fluorescence strength of the enhanced green fluorescent protein through fluorescence-activated cell sorting. The expressions of PTDH1, PPGK1, PINO1, PSED1, and PCCW12 were stronger than that of PTDH3, whereas those of another 15 promoters were stronger than that of PTEF1. Then, 30 promoters were chosen to optimize the biosynthesis pathway of (2S)-naringenin from p-coumaric acid. With a high-throughput screening method, the highest titer of (2S)-naringenin in a 5 L bioreactor reached 1.21 g/L from p-coumaric acid, which is the highest titer according to the currently available reports.

Moisture content is a key factor affecting success of both field seed survival and seed cryobanks. Studying the freezing tolerance of hydrated seeds can provide information for above issues. Our previous study indicated that the ubiquitin-mediated proteolysis pathway was involved in the freezing tolerance of hydrated lettuce seeds. In this study, parthenolide, a repressor of ring-finger-type ubiquitin E3 ligase, was found to significantly reduce the freezing tolerance of lettuce seeds in slow cooling treatment. Differential scanning calorimeter (DSC) proved that parthenolide led to earlier and more ice crystal formation in the embryo of hydrated seeds in slow cooling, compared with control treatment. Real time PCR analysis showed that parthenolide changed the transcription rhythm of the RING-type E3 ligase COP1 and cold-induced gene ICE1 (Inducer of CBF Expression 1). Results of RNA-seq and real time PCR indicated that parthenolide affected the function of endoplasmic reticulum associated degradation (ERAD) pathway, in which Hsp90 was significantly up-regulated and Hsp20 down-regulated after slow cooling. Two-dimensional electrophoresis of total proteins and SDS-PAGE of soluble proteins found that parthenolide inhibited the proteolysis of seed storage globulins in slow cooling treatment. The inhibition of proteolysis for seed storage globulins and the reduction of small molecular chaperons that can prevented aggregation of misfolded proteins in parthenolide-treated seeds may be responsible for the reduction of freezing tolerance of the hydrated seeds. Parthenolide can disrupt the expression pattern of COP1 and cold responsive genes, reduce the level of small molecular chaperons and the proteolysis of seed globulins, which makes hydrated seeds freezing-sensitive.

l -Tyrosine is an important amino acid widely used in food, agriculture, and pharmaceutical industries. However, the industrial application was severely constrained due to low production. To obtain the Escherichia coli mutant producing l -tyrosine in abundance, the heat-inducible expression vector carrying the two feedback resistance enzymes (3-deoxy-7-phosphoheptulonate synthase encoded by aroGfbr and chorismate mutase/prephenate dehydrogenase encoded by tyrAfbr) were introduced into the phenylalanine-producing E. coli strain to enable it to synthesize l -tyrosine directly from glucose. Furthermore, the CRISPR-Cas9 technology was applied to eliminate l -phenylalanine and l -tryptophan pathways for their competition for the carbon flux. The global regulatory protein TyrR, which mediates the biosynthesis and transportation of aromatic amino acids, was also deleted to increase l -tyrosine production. Among the recombinant strains, the pheA/tyrR double-gene deletion strain had the highest yield of 5.84 g/L on shake flasks. The feeding strategies were then optimized in a 3-L fermentor. The pheA/tyrR double-gene deletion strain with the heat-inducible expression plasmid pAP-aroGfbr-tyrAfbr was able to produce 55.54 g/L l -tyrosine by fed-batch fermentation; the substrate conversion rate was 0.25 g/g. The recombinant strains constructed in this study could be an industrial platform for the microbial production of l -tyrosine directly from glucose.

Potassium‐based energy storage technologies, especially potassium ion batteries (PIBs), have received great interest over the past decade. A pivotal challenge facing high‐performance PIBs is to identify advanced electrode materials that can store the large‐radius K+ ions, as well as to tailor the various thermodynamic parameters. Metal chalcogenides are one of the most promising anode materials, having a high theoretical specific capacity, high in‐plane electrical conductivity, and relatively small volume change on charge/discharge. However, the development of metal chalcogenides for PIBs is still in its infancy because of the limited choice of high‐performance electrode materials. However, numerous efforts have been made to conquer this challenge. In this article, we overview potassium storage mechanisms, the technical hurdles, and the optimization strategies for metal chalcogenides and highlight how the adjustment of the crystalline structure and choice of the electrolyte affect the electrochemical performance of metal‐chalcogenide‐based electrode materials. Other potential potassium‐based energy storage systems to which metal chalcogenides can be applied are also discussed. Finally, future research directions focusing on metal chalcogenides for potassium storage are proposed.

Fiber-shaped supercapacitors (FSSCs) show great potential in portable and wearable electronics due to their unique advantages of high safety, environmental friendliness, high performances, outstanding flexibility and integrability. They can directly act as the power sources or be easily integrated with other flexible devices to constitute self-powered and sustainable energy suppliers, providing excellent adaptability to irregular surfaces. This review mainly summarizes the recently reported works of FSSCs including preparation methods of various fiber electrodes, construction strategies of FSSCs and multi-functional device integrations, exploration of reaction mechanisms and strategies to improve the electrochemical performance and provision of suggestions on further designing and optimization of FSSCs. Meanwhile, it shares our perspectives on challenges and opportunities in this field, shedding light on the development of high-performance fiber-shaped supercapacitors with multi-functions.

Background Cultured meat has emerged as a breakthrough technology for the global food industry, which was considered as a potential solution to mitigate serious environmental, sustainability, global public health, and animal welfare concerns in the near future. Although there is promise that cultured meat can supplement or even replace conventional meat, many challenges still need to be resolved in the early stages. Scope and approach In this review, we focused on the characteristics of cultured meat and summarized the current technological challenges and their possible solutions based on tissue and bioreactor engineering, food science, and material science for preparing stem cells, optimizing culture conditions, and developing cost-effective culture media, bioreactor designs, and food processing systems. Key findings and conclusions With rapid progress in tissue and bioreactor engineering, new technologies for culturing meat have been developed and significant progress has been made in recent years. However, as research on cultured meat is intrinsically complex, it is necessary to encourage the integration of multidisciplinary research in this field in the future. We propose some innovative approaches, such as the applications of synthetic biology and bioreactor engineering. These strategies will be helpful to scale-up cultured meat in future applications.

Network state degradation probabilities increase with traffic density. Specifically, at critical or congested densities, any over-input traffic flow may cause degradation, local traffic jams, and even hysteresis of the road network. Existing macroscopic traffic control methods usually target the maximum capacity or minimum delay to improve the road network efficiency and do not consider the negative effect of perimeter control on the state transfer of the road network. This study provides a method to prevent the transfer of the network state when implementing boundary control. First, according to the data detected for the road network, the concept of a conditional value at risk is used to establish a risk decision model that considers the influence of the boundary input flow rate on sub-region state degradation. Then, based on the risk decision model, we propose a state transfer risk decision (STRD) perimeter control method for a large-scale traffic network with multiple sub-regions. This perimeter control method predicts the traffic demand of every sub-region, selects an acceptable traffic flow range at the sub-region boundary by risk interval, regards the maximum trip completion flow and the minimum total travel time as the decision-making objectives, and controls multiple sub-region boundaries. The simulation results show that compared with proportional integral (PI) control and no STRD (NSTRD) control, the STRD control scheme can effectively prevent the state transfer of all sub-regions, improve the trip completion flow, and decrease the travel delay of the network.

China has made great efforts in air pollution control since 2013. However, there is a lack of evaluation of environmental, health and economic co-benefits associated with the national and local air pollution control measures at a city level. We analyzed local air pollution control policies and implementation in Jinan, one of the most heavily air-polluted cities in China between 2013 and 2017. We assessed the changes in exhaust emissions, air quality, mortality and morbidity of associated specific-diseases, and related economic benefits. We also projected the future scenarios of PM2.5 concentration dropped to 15 μg/m3. There were significant decreases in exhaust emissions of SO2 and NOx in Jinan during the study period. Annual reductions in ambient air pollution were 72.6% for SO2, 43.1% for PM2.5, and 34.2% for PM10. A total of 2,317 (95%CI: 1,533–2,842) premature deaths and 15,822 (95%CI: 8,734–23,990) related morbidity cases had been avoided in 2017, leading to a total of US$ 317.7 million (95%CI: 227.5–458.1) in economic benefits. Decreasing PM2.5 concentrations to 15 μg/m3 would result in reductions of 70% in total PM2.5-related non-accidental mortality and 95% in total PM2.5-related morbidity, which translates into US$ 1,289.5 million (95%CI: 825.8–1,673.6) in economic benefits. The national and local air pollution control measures have brought significant environmental, health and economic benefits to a previously heavy polluted Chinese city.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5′-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.

Quasi-solid-state fiber-shaped aqueous supercapacitors (FSASs) are regarded as one of the most promising candidates for high-performance energy storage devices of wearable consumer electronics, due to outstanding cycle stability, high safety and decent mechanical flexibility. The dominated obstacle facing current FSASs system is their quite limited energy density, which is the consequence of insufficient active sites of electrodes, and narrow operating voltage window of electrolyte. In the light of the outlined issues, a novel symmetric FSAS comprising surface-wrinkled and N-doped reduced graphene oxide (rGO) fiber (NRGF) electrodes matched with concentrated “water-in-salt” gel polymer electrolyte (GPE, 8.96 M) was reported in this work. Benefit from the one-dimensional porous microstructure of NRGF and improved hydration energy of GPE, the assembled FSAS has delivered a widely broadened voltage window of 0–2.2 V, an ultrahigh energy density of 25.6 μWh cm−2 (22.7 mWh cm−3) and an outstanding cycling stability for over 20,000 cycles. The electrochemical performance of this FSAS can be well-maintained even under different deformation conditions or after numerous bending cycles. Interestingly, the as-fabricated FSAS can also deliver high energy density from 8.4 μWh cm−2 to 36.6 μWh cm−2 in a temperature range of -20-70 °C, exhibiting a great potential for wide-temperature aqueous energy device.

Nickel-based alloys have been recognized as promising catalysts for upgrading of bio-oils because of their superior deoxygenation activity. Herein, we present a density-functional theory study on hydrodeoxygenation (HDO) of catechol on three Ni-based (111) surfaces, namely Ni(111), bimetallic Fe@Ni(111) single-atom alloy and NiFe(111) homogeneous alloy. The results show that the partial hydrogenation (PH) and/or transhydrogenation (TH) followed by dehydroxylation (DO) are the dominant reaction pathways of catechol HDO towards phenol formation on the three Ni-based (111) surfaces. Specially, TH via *H-and-metal-assisted intramolecular H-transfer is identified as an effective hydrogenation route with a lowered activation barrier compared to the conventional hydrogenation mechanism catalyzed by metals. It is revealed that the deoxygenation performance in catechol HDO can be greatly enhanced by increasing the content of alloyed oxophilic Fe, which, however, could cause the side effect of difficult removal of over-bound *OH on an over-oxophilic surface. The composition of a secondary oxophilic metal in an alloyed surface should be an optimizable parameter as important as altering oxophilic metals alloyed into an active metal phase.

Heparin monitoring is widely used to measure the anticoagulant effect of unfractionated heparin and adjust the dose to keep within the target treatment range. This technology has applications in many fields and also prospects in the future. Its application has the advantages of rapidity, high throughput and minimum sample consumption. Many point of care devices for heparin monitoring are available. The CoaguChek device only requires a small sample size, which is obtained through a fingerstick. Over the last few years, the point-of-care (POC) testing was used widely for its convenience, efficiency, and faster turnaround times.

Flexible metal–gas batteries have become increasingly attractive for use in wearable electronics in the recent years due to their large theoretical energy density and superior adaptability to irregular geometric surfaces, such as the human body. With continuous improvements in design strategies and assembly technologies, the fabrication of various advanced flexible metal–gas batteries has been attempted. In spite of these efforts, the synchronous integration of high flexibility, safety, comfort, and high performance into flexible metal–gas batteries with specifically functionalized configurations still remains a formidable challenge. To resolve these dilemmas, the redesign of cathode catalysts, gel polymer electrolyte, and battery configurations/components has been investigated. In this paper, we review the recent technical advances together with the major dilemmas facing currently available flexible metal–gas batteries, highlighting how flexible cathodes and gel polymer electrolytes with various structures and components can affect the electrochemical performance and functionality of flexible metal–gas batteries. Flexible Zn–air, Li–O2/air, and Li–CO2 batteries are mainly exemplified to elucidate their promising potential. Finally, based on our considerations, unresolved technical hurdles and future research perspectives involving flexible metal–gas batteries for wearable electronics are proposed.

Animal fat as the key component of sensory experience impacts texture, juiciness, and aroma pleasantness of meat, which indicates the necessity of designing fat mimetics in meat alternatives. In this study, high internal phase emulsions (HIPE) with tunable flavor release as fat mimetics based on glycyrrhizic acid (GA) and phytosterol were prepared, and the effects of GA and phytosterol concentrations on the microstructural, rheological, and flavor release properties of HIPE were evaluated. Phytosterol crystals-enriched oil droplets were trapped inside the GA fibrillar matrix as stabilizers. HIPE containing higher GA and phytosterol concentrations exhibited smaller droplet size and better viscoelastic attributes. Additionally, phytosterol played a synergistic role with GA to form a double-fiber microstructure at the oil-water interface. This hierarchical microstructure of oil phase, interface and aqueous phase in the HIPE could regulate the release of hydrophilic and lipophilic meat volatiles. HIPE as fat mimetics with unique microstructure have potential applications in meat alternatives.

Jingjun Huang,1,* Yongjian Guo,1,* Wensou Huang,1,* Xiaotao Hong,2 Yi Quan,3 Liteng Lin,1 Jingwen Zhou,1 Licong Liang,1 Yaqin Zhang,4 Juan Zhou,5 Mingyue Cai,1 Kangshun Zhu1 1Department of Minimally Invasive Interventional Radiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou City, Guangdong Province, People’s Republic of China; 2Department of Interventional Medical Center, Jieyang People’s Hospital, Jieyang City, Guangdong Province, People’s Republic of China; 3Department of Oncology Medical Center, The First People’s Hospital of Zhaoqing, Zhaoqing City, Guangdong Province, People’s Republic of China; 4Department of Radiology, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai City, Guangdong Province, People’s Republic of China; 5Department of Pharmacy, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou City, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Kangshun Zhu; Mingyue Cai, Department of Minimally Invasive Interventional Radiology, the Second Affiliated Hospital of Guangzhou Medical University, 250 East Changgang Road, Guangzhou City, Guangdong, 510260, People’s Republic of China, Tel +86-20-34156205, Fax +86-20-34153709, Email zhksh010@163.com; cai020@yeah.netPurpose: To evaluate the safety and efficacy of regorafenib combined with anti-PD-1 antibody sintilimab (rego-sintilimab) as a second-line treatment for advanced hepatocellular carcinoma (HCC).Methods: This multicenter retrospective study evaluated consecutive patients with advanced HCC who received rego-sintilimab (rego-sintilimab group) or regorafenib alone (regorafenib group) as a second-line treatment from January 2019 to December 2020. Adverse events, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were compared between the two groups. Uni- and multi-variable analyses of prognostic factors for OS and PFS were performed using Cox proportional hazard regression models.Results: In total, 113 patients were included in the study: 58 received rego-sintilimab and 55 received regorafenib. The rego-sintilimab group had higher ORR (36.2% vs 16.4%, P = 0.017), longer PFS (median 5.6 vs 4.0 months; P = 0.045), and better OS (median 13.4 vs 9.9 months; P = 0.023) than the regorafenib group. Regorafenib alone, Child-Pugh B, and neutrophil-to-lymphocyte ratio (NLR) > 3.6 were independent prognostic factors for poor OS. Regorafenib alone, α-fetoprotein level, and NLR > 3.6 were independent prognostic factors for poor PFS. Subgroup analyses showed a survival benefit of rego-sintilimab in patients with NLR ≤ 3.6 (hazard ratio 0.518 [95% CI, 0.257– 0.955]) but not in those with NLR > 3.6 (0.852 [0.461– 1.572]); P = 0.002 for interaction. The difference in incidence of grade 3/4 adverse events between the two groups was not statistically significant (39.7% vs 30.9%; P = 0.331).Conclusion: Rego-sintilimab was tolerated and led to better OS than regorafenib as a second-line treatment for advanced HCC patients, especially in those with NLR ≤ 3.6.Keywords: liver cancer, regorafenib, anti-PD-1 antibody, second-line treatment, overall survival, neutrophil-to-lymphocyte ratio