Progress in research on the biosynthesis of 1,2,4-butanetriol by engineered microbes.

1,2,4-butanetriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT (the R-enantiomer). BT can be used for the synthesis of 1,2,4-butanetriol trinitrate, 3-hydroxytetrahydrofuran, polyurethane, and other chemicals. It is widely used in the military industry, medicine, tobacco, polymer. At present, the BT is mainly synthesized by chemical methods, which are accompanied by harsh reaction conditions, poor selectivity, many by-products, and environmental pollution. Therefore, BT biosynthesis methods with the advantages of mild reaction conditions and green sustainability have become a current research hotspot. In this paper, the research status of microbial synthesis of BT was summarized from the following three aspects: (1) the biosynthetic pathway establishment for BT from xylose; (2) metabolic engineering strategies employed for improving BT production from xylose; (3) other substrates for BT production. Finally, the challenges and prospects of biosynthetic BT were discussed for future methods to improve competitiveness for industrial production.

Characterization of an efficient N-oxygenase from Saccharothrix sp. and its application in the synthesis of azomycin.

BACKGROUND: The nitro group constitutes a significant functional moiety within numerous valuable substances, such as nitroimidazoles, a class of antimicrobial drugs exhibiting broad spectrum activity. Conventional chemical methods for synthesizing nitro compounds suffer from harsh conditions, multiple steps, and environmental issues. Biocatalysis has emerged as a promising alternative to overcome these drawbacks, with certain enzymes capable of catalyzing nitro group formation gradually being discovered in nature. Nevertheless, the practical application is hindered by the restricted diversity and low catalytic activity exhibited by the reported nitrifying enzymes. RESULTS: A novel N-oxygenase SaRohS harboring higher catalytic capability of transformation 2-aminoimidazole to azomycin was characterized from Saccharothrix sp. Phylogenetic tree analysis revealed that SaRohS belongs to the heme-oxygenase-like diiron oxygenase (HDOs) family. SaRohS exhibited optimal activity at pH 5.5 and 25 â„ƒ, respectively. The enzyme maintained relatively stable activity within the pH range of 4.5 to 6.5 and the temperature range of 20 â„ƒ to 35 â„ƒ. Following sequence alignment and structural analysis, several promising amino acid residues were meticulously chosen for catalytic performance evaluation. Site-directed mutations showed that threonine 75 was essential for the catalytic activity. The dual mutant enzyme G95A/K115T exhibited the highest catalytic efficiency, which was approximately 5.8-fold higher than that of the wild-type and 22.3-fold higher than that of the reported N-oxygenase KaRohS from Kitasatospora azatica. The underlying catalytic mechanism was investigated through molecular docking and molecular dynamics. Finally, whole-cell biocatalysis was performed and 2-aminoimidazole could be effectively converted into azomycin with a reaction conversion rate of 42% within 14 h. CONCLUSIONS: An efficient N-oxygenase that catalyzes 2-aminoimidazole to azomycin was screened form Saccharothrix sp., its phylogenetics and enzymatic properties were analyzed. Through site-directed mutation, enhancements in catalytic competence were achieved, and the molecular basis underlying the enhanced enzymatic activity of the mutants was revealed via molecular docking and dynamic simulation. Furthermore, the application potential of this enzyme was assessed through whole cell biocatalysis, demonstrating it as a promising alternative method for azomycin production.

Highly Transparent, Temperature-Resistant, and Flexible Polyimide Aerogels for Solar Energy Collection.

Advanced aerogel materials with low thermal conductivity and high transparency have shown great application prospects in the solar thermal energy conversion field. However, most aerogels do not meet these requirements due to their low optical transparency and poor mechanical properties. To tackle this problem, we have created versatile polyimide (PI) aerogel materials by adjusting the monomers to alter their molecular structure. These materials exhibit exceptional thermal insulation properties and high transparency, making them ideal for use in the construction of efficient solar collector devices. Incorporating 1,3,5-benzenetricarbonyl trichloride into PI aerogel results in high strength (>3 MPa) and excellent transmittance (>90%) over a broad range of wavelengths (500-2650 nm). The as-prepared PI aerogel solar collector (PIASC) also exhibits a low thermal conductivity (0.032 W/mK), a low density (0.1 g/cm3), and high porosity (90%). By changing the shape of the collector from a flat plate to a cylindrical ring, the heat collection efficiency and capacity are significantly improved, resulting in efficient heat collection. The circular ring collector has a maximum heat collection temperature of 236.8 °C. The PIASC, which is both flexible and highly transparent, is an ideal candidate for advanced optical elements and solar collectors.

Biosynthesis of (R)-(+)-perillyl alcohol by Escherichia coli expressing neryl pyrophosphate synthase.

(R)-(+)-perillyl alcohol is widely used in agricultural and anticarcinogenic fields. Microbial production of (R)-(+)-perillyl alcohol was investigated in this study. We optimized biosynthesis of (R)-(+)-perillyl alcohol in Escherichia coli by using neryl pyrophosphate synthase and NADPH regeneration. Engineering neryl pyrophosphate (NPP)-supplied pathway resulted in a 4-fold improvement of (R)-(+)-perillyl alcohol titer. Subsequently, combined engineering of p-cymene monooxygenase (CymA) expression and module for NADPH regeneration exhibited a 15.4-fold increase of titer over the initial strain S02. Finally, 453 mg/L (R)-(+)-perillyl alcohol was achieved in fed-batch fermentation, which is the highest (R)-(+)-perillyl alcohol titer in E. coli.

Efficient Synthesis of (R)-(+)-Perillyl Alcohol From (R)-(+)-Limonene Using Engineered Escherichia coli Whole Cell Biocatalyst.

(R)-(+)-perillyl alcohol is a much valued supplemental compound with a wide range of agricultural and pharmacological characteristics. The aim of this study was to improve (R)-(+)-perillyl alcohol production using a whole-cell catalytic formula. In this study, we employed plasmids with varying copy numbers to identify an appropriate strain, strain 03. We demonstrated that low levels of alKL provided maximal biocatalyst stability. Upon determination of the optimal conditions, the (R)-(+)-perillyl alcohol yield reached 130 mg/L. For cofactor regeneration, we constructed strain 10, expressing FDH from Candida boidinii, and achieved (R)-(+)-perillyl alcohol production of 230 mg/L. As a result, 1.23 g/L (R)-(+)-perillyl alcohol was transformed in a 5 L fermenter. Our proposed method facilitates an alternative approach to the economical biosynthesis of (R)-(+)-perillyl alcohol.

Highly efficient biosynthesis of ß-caryophyllene with a new sesquiterpene synthase from tobacco.

BACKGROUND: ß-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids. RESULTS: A new gene of ß-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L ß-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing ß-caryophyllene production to 100.3 mg/L. The highest production of ß-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of ß-caryophyllene by 20% to 5142 mg/L. CONCLUSION: A new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce ß-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of ß-caryophyllene than the previous studies. During the fermentation process, we observed that ß-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.

Recent advances in the biosynthesis of isoprenoids in engineered Saccharomyces cerevisiae.

Isoprenoids, as the largest group of chemicals in the domains of life, constitute more than 50,000 members. These compounds consist of different numbers of isoprene units (C5H8), by which they are typically classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids (C40). In recent years, isoprenoids have been employed as food additives, in the pharmaceutical industry, as advanced biofuels, and so on. To realize the sufficient and efficient production of valuable isoprenoids on an industrial scale, fermentation using engineered microorganisms is a promising strategy compared to traditional plant extraction and chemical synthesis. Due to the advantages of mature genetic manipulation, robustness and applicability to industrial bioprocesses, Saccharomyces cerevisiae has become an attractive microbial host for biochemical production, including that of various isoprenoids. In this review, we summarized the advances in the biosynthesis of isoprenoids in engineered S. cerevisiae over several decades, including synthetic pathway engineering, microbial host engineering, and central carbon pathway engineering. Furthermore, the challenges and corresponding strategies towards improving isoprenoid production in engineered S. cerevisiae were also summarized. Finally, suggestions and directions for isoprenoid production in engineered S. cerevisiae in the future are discussed.

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol.

BACKGROUND: (R)-(+)-perillyl alcohol is a naturally oxygenated monoterpene widely used as the natural flavor additives, insecticides, jet fuels and anti-cancer therapies. It was also readily available monoterpene precursors. However, this natural product is present at low concentrations from plant sources which are not economically viable. Therefore, alternative microbial production methods are rapidly emerging as an attractive alternative to make (R)-(+)-perillyl alcohol production more sustainable and environmentally friendly. RESULTS: We engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase, P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of (R)-(+)-perillyl alcohol. The concentration of (R)-(+)-limonene (the monoterpene precursor to (R)-(+)-perillyl alcohol) reached 45 mg/L from glucose. Enhanced (R)-(+)-perillyl alcohol production was therefore achieved. The strain produced (R)-(+)-perillyl alcohol at a titer of 87 mg/L and a yield of 1.5 mg/g glucose in a 5 L bioreactor fed batch system. CONCLUSIONS: These datas highlight the efficient production of (R)-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.

Metabolic Engineering of Different Microbial Hosts for Lycopene Production.

As a result of the extensive use of lycopene in a variety of fields, especially the dietary supplement and health food industries, the production of lycopene has attracted considerable interest. Lycopene can be obtained through extraction from vegetables and chemical synthesis. Alternatively, the microbial production of lycopene has been extensively researched in recent years. Various types of microbial hosts have been evaluated for their potential to accumulate a high level of lycopene. Metabolic engineering of the hosts and optimization of culture conditions are performed to enhance lycopene production. After years of research, great progress has been made in lycopene production. In this review, strategies used to improve lycopene production in different microbial hosts and the advantages and disadvantages of each microbial host are summarized. In addition, future perspectives of lycopene production in different microbial hosts are discussed.

Associations of procalcitonin, C-reaction protein and neutrophil-to-lymphocyte ratio with mortality in hospitalized COVID-19 patients in China.

Coronavirus disease 2019 (COVID-19) is an important and urgent threat to global health. Inflammation factors are important for COVID-19 mortality, and we aim to explore whether the baseline levels of procalcitonin (PCT), C-reaction protein (CRP) and neutrophil-to-lymphocyte ratio (NLR) are associated with an increased risk of mortality in patients with COVID-19. A retrospective study was conducted and a total of 76 patients with confirmed COVID-19 were included between January 17, 2020 to March 2, 2020, of these cases, 17 patients were dead. After adjusting covariates, PCT (≥ 0.10 ng/mL) and CRP (≥ 52.14 mg/L) exhibited independent increasing risks of mortality were used hazard ratio (HR) of 52.68 (95% confidence interval [CI]: 1.77-1571.66) and 5.47 (95% CI: 1.04-28.72), respectively. However, NRL (≥ 3.59) was not found to be an independent risk factor for death in our study. Furthermore, the elevated PCT levels were still associated with increasing risk of mortality in the old age group (age ≥ 60 y), and in the critically severe and severe patients after adjustment for complications. Thu Baseline levels of PCT and CRP have been addressed as independent predictors of mortality in patients with COVID-19.

Biosynthesis of 2,4-diacetylphloroglucinol from glucose using engineered Escherichia coli.

In order to produce 2,4-diacetylphloroglucinol (2,4-DAPG) in E. coli, the key synthases coding by phlACBD gene cluster from the strain Pseudomonas fluorescens CHA0 were overexpressed in E. coli BL21 (DE3). The marA, phlE and acc genes were also overexpressed to enhance 2,4-DAPG biosynthesis. Then the fermentation conditions were optimized to improve the concentration of 2,4-DAPG. The results showed that the recombinant E. coli could produce few 2,4-DAPG with only the phlACBD gene cluster. The synthetic ability of 2,4-DAPG could be increased by expressing the acc, marA and phlE genes in shake-flasks cultivation. The effects of phloroglucinol, initial pH, temperature and trace elements on 2,4-DAPG biosynthesis were also investigated. Based on the optimal fermentation conditions obtained from the shake-flasks cultivation, fed-batch fermentation of strain Z3 in a 5 L bioreactor was conducted to produce 2,4-DAPG. Finally, the concentration of 2,4-DAPG was 179 mg/L after induction for 36 h by fed-batch fermentation. To the best of our knowledge, this is the highest 2,4-DAPG production reported in E. coli. This work showed the potential application of engineered E. coli to get high production of target compounds.

Effect of grelin on TRX expression in chronic heart failure tissue: A protocol of systematic review and meta-analysis.

BACKGROUND: The aim of this study is to explore the effect of grelin on TRX expression (TRXE) in chronic heart failure tissue (CHFT). METHODS: We will search electronic databases from inception to the March 1, 2020 in MEDLINE, EMBASE, Cochrane Library, CINAHL, PEDro, the Allied and Complementary Medicine Database, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure. We will not apply any limitations to the language and publication status. Any randomized controlled trials (RCTs) that studied the effect of grelin on TRXE in CHFT will be included. Study quality will be checked by Cochrane risk of bias and evidence quality will be appraised by Grading of Recommendations Assessment Development and Evaluation. All extracted data will be analyzed by RevMan 5.3 Software. RESULTS: This study will summarize the present RCTs to assess the effect of grelin on TRXE in CHFT. CONCLUSION: The results of this study will provide conclusive evidence of the effect of grelin on TRXE in CHFT. SYSTEMATIC REVIEW REGISTRATION: INPLASY202040078.

An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure ß-Phenylalanine.

An aminotransferase ω-TAEn was identified from Enhydrobacter aerosaccus. The ω-TAEn was successfully expressed in Escherichia coli and the obtained enzyme showed activity toward ß-phenylalanine (ß-phe) at optimal conditions. For optically pure (R)-ß-phe, 50% yield was observed by kinetic resolution of racemic amino with pyruvate as the amino acceptor. To obtain (S)-ß-phe, the lipase/ω-TAEn catalytic system was adopted. The ω-TAEn showed strict stereoselectivity to the amino donor. The formation of (S)-ß-phe was observed using 3-aminobutyric acid as the amino donor, and (S)-ß-phe was obtained by asymmetric synthesis with a yield of 82%.

Metabolic engineering of Escherichia coli for the utilization of ethanol.

BACKGROUND: The fuel ethanol industry has made tremendous progress in the last decades. Ethanol can be obtained by fermentation using a variety of biomass materials as the feedstocks. However, few studies have been conducted on ethanol utilization by microorganisms. The price of petroleum-derived ethanol, easily made by the hydrolysis of ethylene, is even lower than that of bioethanol. If ethanol can be metabolized by microorganisms to produce value-added chemicals, it will open a new door for the utilization of inexpensive ethanol resources. RESULTS: We constructed an engineered Escherichia coli strain which could utilize ethanol as the sole carbon source. The alcohol dehydrogenase and aldehyde dehydrogenase from Aspergillus nidulans was introduced into E. coli and the recombinant strain acquired the ability to grow on ethanol. Cell growth continued when ethanol was supplied after glucose starvation and 2.24 g L-1 of ethanol was further consumed during the shake-flasks fermentation process. Then ethanol was further used for the production of mevalonic acid by heterologously expressing its biosynthetic pathway. Deuterium-labeled ethanol-D6 as the feedstock confirmed that mevalonic acid was synthesized from ethanol. CONCLUSIONS: This study demonstrated the possibility of using ethanol as the carbon source by engineered E. coli strains. It can serve as the basis for the construction of more robust strains in the future though the catabolic capacity of ethanol should be further improved.

Increasing the pyruvate pool by overexpressing phosphoenolpyruvate carboxykinase or triosephosphate isomerase enhances phloroglucinol production in Escherichia coli.

OBJECTIVES: Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. RESULTS: In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. CONCLUSIONS: These results indicate that overexpression of pckA or tpiA can enhance the pyruvate "pool" and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.

Electricigens in the anode of microbial fuel cells: pure cultures versus mixed communities.

Microbial fuel cell (MFC) is an environmentally friendly technology for electricity harvesting from a variety of substrates. Microorganisms used as catalysts in the anodic chamber, which are termed as electricigens, play a major role in the operation of MFCs. This review provides an introduction to the currently identified electricigens on their taxonomical groups and electricity producing abilities. The mechanism of electron transfer from electricigens to electrode is highlighted. The performances of pure culture and mixed communities are compared particularly. It has been proved that the electricity generation capacity and the ability to adapt to the complex environment of MFC systems constructed by pure microbial cultures are less than the systems constructed by miscellaneous consortia. However, pure cultures are useful to clarify the electron transfer mechanism at the microbiological level and further reduce the complexity of mixed communities. Future research trends of electricigens in MFCs should be focused on screening, domestication, modification and optimization of multi-strains to improve their electrochemical activities. Although the MFC techniques have been greatly advanced during the past few years, the present state of this technology still requires to be combined with other processes for cost reduction.

Improving the production of isoprene and 1,3-propanediol by metabolically engineered Escherichia coli through recycling redox cofactor between the dual pathways.

The biosynthesis of isoprene by microorganisms is a promising green route. However, the yield of isoprene is limited due to the generation of excess NAD(P)H via the mevalonate (MVA) pathway, which converts more glucose into CO2 or undesired reduced by-products. The production of 1,3-propanediol (1,3-PDO) from glycerol is a typical NAD(P)H-consuming process, which restricts 1,3-PDO yield to ~ 0.7 mol/mol. In this study, we propose a strategy of redox cofactor balance by coupling the production of isoprene with 1,3-PDO fermentation. With the introduction and optimization of the dual pathways in an engineered Escherichia coli, ~ 85.2% of the excess NADPH from isoprene pathway was recycled for 1,3-PDO production. The best strain G05 simultaneously produced 665.2 mg/L isoprene and 2532.1 mg/L 1,3-PDO under flask fermentation conditions. The yields were 0.3 mol/mol glucose and 1.0 mol/mol glycerol, respectively, showing 3.3- and 4.3-fold improvements relative to either pathway independently. Since isoprene is a volatile organic compound (VOC) whereas 1,3-PDO is separated from the fermentation broth, their coproduction process does not increase the complexity or cost for the separation from each other. Hence, the presented strategy will be especially useful for developing efficient biocatalysts for other biofuels and biochemicals, which are driven by cofactor concentrations.

Manipulation of the precursor supply for high-level production of longifolene by metabolically engineered Escherichia coli.

Longifolene is a naturally occurring tricyclic sesquiterpene widely used in many different fields. Up to now, this valuable terpene was mainly manufactured from the high-boiling fraction of certain pine resins. Microbial production can be a promising alternative to the extraction from natural plant sources. Here, we present the metabolic engineering strategy to assemble biosynthetic pathway for longifolene production in Escherichia coli. E. coli was rendered to produce longifolene by heterologously expressing a codon optimized longifolene synthase from Picea abies. Augmentation of the metabolic flux to farnesyl pyrophosphate (FPP) by different FPP synthases conferred a 1.8-fold increase in longifolene production. An additional enhancement of longifolene production (up to 2.64 mg/L) was achieved by introducing an exogenous mevalonate pathway. Under fed-batch conditions, the best-performing strain was able to produce 382 mg/L of longifolene in a 5 L bioreactor. These results demonstrated the feasibility of producing longifolene by microbial fermentation and could serve as the basis for the construction of more robust strains in the future.

[Perillyl alcohol production by engineered heterologous mevalonate pathway in Escherichia coli].

Perillyl alcohol, [4-isopropylene-1-cyclohexene] methanol, is a monocyclic monoterpene alcohol with special odorous similar to that of linalool and terpineol. It has application potential in pharmaceutical, daily chemical and food industries. In this study, one method for the synthesis of perillyl alcohol through the MVA pathway was studied. First, the MVA metabolic pathway originated from Enterococcus faecalis was constructed in Escherichia coli to synthesize limonene. Limonene was further transformed to perillyl alcohol by the hydroxylation of cytochrome P450 alkane hydroxylase. Furthermore, the shake flask fermentation condition of the engineered E. coli strain was optimized. The results showed that the engineered E. coli could produce about 50.12 mg/L perillyl alcohol through MVA pathway using glucose as raw material. In this study, the method of the MVA pathway for perillyl alcohol synthesis was constructed successfully in engineered E. coli, which provides both theoretical and technical support for terpenoids biosynthesis.

Biosynthesis and production of sabinene: current state and perspectives.

Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production.