Simple Carbon Sources Research Articles

Simple carbon and organic matter addition in acid sulfate soils and time-dependent changes in pH and redox under varying moisture regimes

Abstract. Michael PS. 2020. Simple carbon and organic matter addition in acid sulfate soils and time-dependent changes in pH and redox under varying moisture regimes. Asian J Agric 4: 23-29. It has been recently shown that the addition of dead plant materials as organic matter in acid sulfate soils (ASS) creates microenvironments conducive for soil microbes to reduce the sulfate content and redox potential (Eh), thereby increasing the pH of sulfuric soil and sustaining the pH of sulfidic soil, respectively. The time course of effects of addition of organic matter however was not clearly established. This was study conducted to assess the time course of the effect of soil carbon and organic matter following addition of glucose and acetate as carbon and lucerne hay as organic matter sources, respectively. The conditions of the treatments were either anaerobic (flooded) or aerobic (under 75% field capacity). The results showed that the effects of addition of amendments on pH and Eh of ASS are immediate, but treatment dependent. Organic matter as a multiple food sources for soil microbes was more effective in reducing the soil and increasing the pH of the sulfuric and the sulfidic soil, respectively. The effects of acetate were comparatively higher than glucose, dependent on the type of microbial ecology that was engaged by these resources being simple carbon sources. The overall increase in pH and reduction in Eh was immediate within the first three days, but the changes in the soil properties measured were reversed over time. The reversal in the mechanism inducing the changes in pH and Eh ceased as the microbial metabolic resources used as metabolic substrate to generate alkalinity got depleted.

Towards Predicting Gut Microbial Metabolism: Integration of Flux Balance Analysis and Untargeted Metabolomics.

Genomics-based metabolic models of microorganisms currently have no easy way of corroborating predicted biomass with the actual metabolites being produced. This study uses untargeted mass spectrometry-based metabolomics data to generate a list of accurate metabolite masses produced from the human commensal bacteria Citrobacter sedlakii grown in the presence of a simple glucose carbon source. A genomics-based flux balance metabolic model of this bacterium was previously generated using the bioinformatics tool PyFBA and phenotypic growth curve data. The high-resolution mass spectrometry data obtained through timed metabolic extractions were integrated with the predicted metabolic model through a program called MS_FBA. This program correlated untargeted metabolomics features from C. sedlakii with 218 of the 699 metabolites in the model using an exact mass match, with 51 metabolites further confirmed using predicted isotope ratios. Over 1400 metabolites were matched with additional metabolites in the ModelSEED database, indicating the need to incorporate more specific gene annotations into the predictive model through metabolomics-guided gap filling.

Carbon metabolic profiling of Trichoderma strains provides insight into potential ecological niches

ABSTRACTMany Trichoderma species are cosmopolitan and widespread free-living fungi in various ecological environment, and many are economically important in the fields of agriculture and industry. However, carbon metabolism profiles of Trichoderma species have not been characterized in detail. In this study, Biolog FF MicroPlates were used to contrast carbon utilization and the differences among five Trichoderma species, each representing a unique phenotype. Their metabolic abilities varied greatly. Species producing cellulases and chitinases and with phosphate-solubilizing activities exhibited high efficiency of substrate utilization, whereas low efficiency was shown by saline-alkaline-tolerant species that metabolized simple carbon sources. Species producing cellulases at high levels may be specified to decompose and assimilate cellulose and hemicellulose in woody substrates, and those producing chitinases may have mycoparasitic roles. Species with plant growth–promoting traits are good at uptake of exudates from plant roots. Overall, metabolic models reflect nutritional adaptation of Trichoderma to diverse niches in nature.

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

Background(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield.ResultsAs the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively.ConclusionWe successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

Putative metabolic pathway for the bioproduction of bikaverin and intermediates thereof in the wild Fusarium oxysporum LCP531 strain

Fungal naphthoquinones, like red bikaverin, are of interest due to their growing applications in designing pharmaceutical products. Though considerable work has been done on the elucidation of bikaverin biosynthesis pathway in Fusarium fujikuroi, very few reports are available regarding its bioproduction in F. oxysporum. We are hereby proposing a putative metabolic pathway for bikaverin bioproduction in a wild F. oxysporum strain by cross-linking the pigment profiles we obtained under two different fermentation conditions with literature. Naphthoquinone pigments were extracted with a pressurized liquid extraction method, and characterized by HPLC–DAD and UHPLC-HRMS. The results led to the conclusions that the F. oxysporum LCP531 strain was able to produce bikaverin and its various intermediates, e.g., pre-bikaverin, oxo-pre-bikaverin, dinor-bikaverin, me-oxo-pre-bikaverin, and nor-bikaverin, in submerged cultures in various proportions. To our knowledge, this is the first report of the isolation of these five bikaverin intermediates from F. oxysporum cultures, providing us with steady clues for confirming a bikaverin metabolic pathway as well as some of its regulatory patterns in the F. oxysporum LCP531 strain, based on the previously reported model in F. fujikuroi. Interestingly, norbikaverin accumulated along with bikaverin in mycelial cells when the strain grew on simple carbon and nitrogen sources and additional cofactors. Along bikaverin production, we were able to describe the excretion of the toxin beauvericin as main extrolite exclusively in liquid medium containing complex nitrogen and carbon sources, as well as the isolation of ergosterol derivate in mycelial extracts, which have potential for pharmaceutical uses. Therefore, culture conditions were also concluded to trigger some specific biosynthetic route favoring various metabolites of interest. Such observation is of great significance for selective production of pigments and/or prevention of occurrence of others (aka mycotoxins).

De novo biosynthesis of indole-3-ethanol and indole-3-ethanol acetate in engineered Escherichia coli

Indole-3-ethanol is an important pharmaceutical intermediate that possess significant pharmaceutical properties. In this study, we established an artificial pathway in engineered Escherichia coli for microbial production of indole-3-ethanol from simple carbon sources. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to indole-3-ethanol. We have further demonstrated the biosynthesis of indole-3-ethanol acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent conversion of indole-3-ethanol to indole-3-ethanol acetate. To our knowledge, this is the first report on the biosynthesis of indole-3-ethanol and indole-3-ethanol acetate directly from a renewable carbon source.

Production of Melanins With Recombinant Microorganisms.

The melanins constitute a diverse group of natural products found in most organisms, having functions related to protection against chemical and physical stresses. These products originate from the enzyme-catalyzed oxidation of phenolic and indolic substrates that polymerize to yield melanins, which include eumelanin, pheomelanin, pyomelanin, and the allomelanins. The enzymes involved in melanin formation belong mainly to the tyrosinase and laccase protein families. The melanins are polymeric materials having applications in the pharmaceutical, cosmetic, optical, and electronic industries. The biotechnological production of these polymers is an attractive alternative to obtaining them by extraction from plant or animal material, where they are present at low concentrations. Several species of microorganisms have been identified as having a natural melanogenic capacity. The development and optimization of culture conditions with these organisms has resulted in processes for generating melanins. These processes are based on the conversion of melanin precursors present in the culture medium to the corresponding polymers. With the application of genetic engineering techniques, it has become possible to overexpress genes encoding enzymes involved in melanin formation, mostly tyrosinases, leading to an improvement in the productivity of melanogenic organisms, as well as allowing the generation of novel recombinant microbial strains that can produce diverse types of melanins. Furthermore, the metabolic engineering of microbial hosts by modifying pathways related to the supply of melanogenic precursors has resulted in strains with the capacity of performing the total synthesis of melanins from simple carbon sources in the scale of grams. In this review, the latest advances toward the generation of recombinant melanin production strains and production processes are summarized and discussed.

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.

Polyhydroxyalkanoates Synthesized by Aeromonas Species: Trends and Challenges.

The negative effects of petrochemical-derived plastics on the global environment and depletion of global fossil fuel supplies have paved the way for exploring new technologies for the production of bioplastics. Polyhydroxyalkanoates (PHAs) are considered an alternative for synthetic polymers because of their biodegradability, biocompatibility, and non-toxicity. Many bacteria have been reported to have the ability to synthesize PHAs. Among them, the Aeromonas species seem to be ideal hosts for the industrial production of these biopolyesters due to their robust growth, simple growth requirements, their ability for the synthesis of homopolymers, co-polymers, and terpolymers with unique material properties. Some Aeromonas strains were able to produce PHAs in satisfactory amounts from simple carbon sources. Efforts have been made to use genetically modified Aeromonas strains for enhanced PHAs and to obtain bacteria with modified compositions and improved properties. This review discusses the current state of knowledge of polyhydroxyalkanoates synthesized by Aeromonas species, with a special focus on their potential, challenges, and progress in PHA synthesis.

Evaluation of Methyl Red and Lactate as a Mediator and a Simple Carbon Source on Electrochemical Performance of Urmia lake Sediment Microbial Fuel Cell

مقدمه: بحران انرژی به‌علت مصرف روزافزون سوخت‌های فسیلی موضوع بسیار مهمی است؛ ازاین‌رو، منابع انرژی جایگزین دارای اهمیت فوق‌العاده‌ای هستند. از میان منابع انرژی تجدیدپذیر، فناوری پیل‌های سوختی میکروبی رسوبی بسیار مهم است؛ در این پیل‌ها با عملکرد کاتالیزوری باکتری‌های مناسب (اگزوالکتروژن[i]) انرژی شیمیایی مواد آلی به انرژی الکتریکی تبدیل می‌شود. مواد و روش‏‏ها: در مطالعۀ حاضر، از ظروف شیشه‌ای یک لیتری به‌عنوان راکتور استفاده شد. نصف ظرف با لجن دریاچۀ ارومیه (بخش آندی) و بخش بالایی ظرف با آب دریاچۀ ارومیه (بخش کاتدی) تعبیه شد. صفحه‌های گرافیتی با مساحت سطح کاربردی 4×4 سانتی‌مترمربع (الکترود انتخابی) به سیم‌های مسی اتصال یافتند و دو بخش متأثر از مقاومت بیرونی 2/2 کیلواهم به‌ هم متصل شدند. عملکرد الکتروشیمیایی پیل‌های سوختی با استفاده از ولت‌‌متر دیجیتالی ارزیابی شد. تأثیر متیل‌رد (واسطۀ انتقال الکترون) و لاکتات (منبع سادۀ کربنی) پس‌از تعیین غلظت بهینه در سه بازۀ 15‌روزه بررسی شد. نتایج: نتایج نشان دادند عملکرد الکتروشیمیایی پیل سوختی میکروبی رسوبی دارای متیل‌رد با دانسیتۀ توان[ii] 54/7 میلی‌وات بر متر‌مربع سطح کاربردی الکترود آندی اختلاف معناداری با پیل سوختی بدون واسطه با دانسیتۀ توان 46/0 میلی‌وات بر متر‌مربع دارد. در مقابل، پیل حاوی متیل‌رد و لاکتات دانسیتۀ توان 41/1±44/4 میلی‌وات بر متر‌مربع را نشان داد. بحث و نتیجه‏گیری: کنسرسیوم میکروبی رسوبات الکترون‌های تولیدشده از تجزیۀ منابع آلی را به‌طور مستقیم در قالب تشکیل بیوفیلم روی الکترود آندی و یا به کمک واسطه‌های سنتتیک به الکترود آندی منتقل می‌کنند. نتایج نشان دادند افزودن واسطۀ سنتتیک خارجی متیل‌رد باعث افزایش عملکرد الکتروشیمیایی پیل سوختی می‌شود. انتظار می‌رفت پیل سوختی عملکرد بهتری در حضور منبع کربن خارجی و متیل‌رد داشته باشد، اما پیل سوختی در نبود لاکتات عملکرد بهتری نشان داد. [i]- Exoelectrogen [ii]- Power density

Understanding metabolic processes shaping adaptation of E. coli to the gut

Many microbes colonise the gut establishing interactions with their host and their nutritional environment. Studying genetics and metabolism brought about the drive and potential to engineer communities to promote health and improve industrial processes. However, structuring artificial communities in predictable ways is underdeveloped. We studied Escherichia coli’s genetic targets and physiological mechanisms during gut colonisation and adaptation and how metabolic environment/microbiota complexity shape these processes. We introduced a tractable E. coli K-12 in mice Germ-free or with polymicrobial communities. Whole Genome Sequencing identified potential adaptive targets. Here, we established phenotypic assays as well characterising effects of key mutations and metabolomics was performed with 1H-NMR of intestinal contents. Genes for sugar alcohol metabolism (gat) was the only target common to both mouse models, evidencing specificity. Facing complex microbiota E. coli targeted use of sugar alcohols (srlR, kdgR) and anaerobic respiration (dcuB, focA) [1] whereas alone, we observed instead mutations pointing to increased ability for amino acid use (lrp, dtpB, alaA). Mutations selected correlated dinamically with metabolomics: our results fit the model whereby other microbiota members scavenge oxygen and breakdown complex sugars, limiting E. coli to anaerobically respire simple by-product carbon sources. In the opposing scenario (functional absence) improved amino acid use are favoured colonisation factors. Through experimental evolution we gained insight on shaping E. coli’s metabolic traits through genetic engineering to colonise specific host environments. This work also highlights the versatility of E. coli as potential biotic sensor. [1] Barroso-Batista, J. et al. The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps, 2014.

Influence of Organic Carbon Sources on Metal Removal from Mine Impacted Water Using Sulfate-Reducing Bacteria Bioreactors in Cold Climates

Acid mine drainage (AMD) is a major environmental challenge for the mining industry in northern climates. Laboratory-scale experiments were conducted to test various simple and complex carbon sources available in the Yukon as electron donors for sulfate reduction to allow the subsequent removal of Cd and Zn from a Yukon AMD. The 1 L capacity bioreactors were monitored biweekly at 5 °C. After 162 days, a diminution of both total organic carbon and sulfate concentrations was observed in all bioreactors. A long adaptation period was necessary before consumption of the carbon source started, which might be due to the cold temperature. Simple organic sources of carbon (methanol and ethylene glycol) and complex organic sources of carbon (potato oil, brewery residue, peat, and straw) were used to support SRB growth. Methanol and ethylene glycol led to a diminution of sulfate concentrations of 71.2 and 36.9%, respectively, while the decrease of sulfate concentrations was limited to 13.8 and 5.3% when using peat and straw, respectively.

The Dynamic of a River Model Bacterial Community in Two Different Media Reveals a Divergent Succession and an Enhanced Growth of Most Strains Compared to Monocultures.

The dynamic of a community of 20 bacterial strains isolated from river water was followed in R2 broth and in autoclaved river water medium for 27days in batch experiments. At an early stage of incubation, a fast-growing specialist strain, Acinetobater sp., dominated the community in both media. Later on, the community composition in both media diverged but was highly reproducible across replicates. In R2, several strains previously reported to degrade multiple simple carbon sources prevailed. In autoclaved river water, the community was more even and became dominated by several strains growing faster or exclusively in that medium. Those strains have been reported in the literature to degrade complex compounds. Their growth rate in the community was 1.5- to 7-fold greater than that observed in monoculture. Furthermore, those strains developed simultaneously in the community. Together, our results suggest the existence of cooperative interactions within the community incubated in autoclaved river water.

Stepwise increase in the production of 13R-manoyl oxide through metabolic engineering of Saccharomyces cerevisiae

Forskolin, a labdane diterpenoid, possesses a wide range of pharmacological activities. (13R)-manoyl oxide (13R-MO) is the precursor of forskolin. As forskolin is a structurally complex, highly oxidized compound, chemical synthesis is tedious and difficult. Herein, we present a biosynthesis method involving metabolic engineering of Saccharomyces cerevisiae to produce 13R-MO at an initial titer of 2.3 mg/l. We further optimized the entire MVA pathway, which increased the FPP supply pool, but resulted in a sharp decrease in the 13R-MO production due to low metabolic flux toward geran ylgeranyl pyrophosphate (GGPP). To mitigate this, we down-regulated the competing pathway by replacing the original promoter of the squalene synthase gene ERG9 with the MET3 promoter, significantly improving the 13R-MO production to 45.2 mg/l. Finally, three feed strategies were investigated in fed-batch fermentation; the glucose feeding strategy enabled the engineered yeast to produce 167.1 ± 5.2 mg/l 13R-MO, equivalent to 2.6 ± 0.08 mg/l/OD600. Here, we describe a systematic synthetic biology method to produce 13R-MO from a simple carbon source using Saccharomyces cerevisiae. This not only enabled a relatively high level of 13R-MO production but also provided an efficient platform for the production of other diterpenoids.

Enhanced amylase production by a Bacillus subtilis strain under blue light-emitting diodes

A chemotrophic, aerobic bacterial strain, Bacillus subtilis B2, was used to produce amylase by submerged fermentation under different light sources. SDS-PAGE indicated that the 55 kDa enzyme belonged to the α-amylase group. B2 was incubated in basal media with 1% soluble starch (pH 7.0) under blue, green, red, and white light-emitting diodes (LEDs), and white fluorescent light. Fermentation under blue LEDs maximized amylase production (180.59 ± 1.6 U/mL at 24 h). Production at 48 h increased to 310.56 ± 1.6 U/mL with 5% glucose as a simple carbon source and to 300.51 ± 1.7 U/mL with 5% groundnut oil cake as an agricultural waste substrate. Activity and stability of the amylase were greatest at pH 7.0 and 45–55 °C. Na+, Ca2+, Mg2+, Co2+, Ba2+, and K+ increased activity, while Ni2+, Hg2+, Mn2+, Cu2+, Fe3+, and Zn2+ inhibited activity. EDTA, PMSF and DTNB reduced activity by 50% or more, while tetrafluoroethylene and 1,10-phenanthroline reduced activity by 30%. The amylase was highly tolerant of the surfactants, compatible with organic solvents, oxidizing agents and the reducing agents reduced activity. These properties suggest utility of amylase produced by B. subtilis B2 under blue LED-mediated fermentation for industrial applications.

Soil Particles and Phenanthrene Interact in Defining the Metabolic Profile of Pseudomonas putida G7: A Vibrational Spectroscopy Approach.

In soil, organic matter and mineral particles (soil particles; SPs) strongly influence the bio-available fraction of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), and the metabolic activity of bacteria. However, the effect of SPs as well as comparative approaches to discriminate the metabolic responses to PAHs from those to simple carbon sources are seldom considered in mineralization experiments, limiting our knowledge concerning the dynamics of contaminants in soil. In this study, the metabolic profile of a model PAH-degrading bacterium, Pseudomonas putida G7, grown in the absence and presence of different SPs (i.e., sand, clays and humic acids), using either phenanthrene or glucose as the sole carbon and energy source, was characterized using vibrational spectroscopy (i.e., FT-Raman and FT-IR spectroscopy) and multivariate classification analysis (i.e., PLS-DA). The different type of SPs specifically altered the metabolic profile of P. putida, especially in combination with phenanthrene. In comparison to the cells grown in the absence of SPs, sand induced no remarkable change in the metabolic profile of the cells, whereas clays and humic acids affected it the most, as revealed by the higher discriminative accuracy (R2, RMSEP and sensitivity) of the PLS-DA for those conditions. With respect to the carbon-source (phenanthrene vs. glucose), no effect on the metabolic profile was evident in the absence of SPs or in the presence of sand. On the other hand, with clays and humic acids, more pronounced spectral clusters between cells grown on glucose or on phenanthrene were evident, suggesting that these SPs modify the way cells access and metabolize PAHs. The macromolecular changes regarded mainly protein secondary structures (a shift from α-helices to β-sheets), amino acid levels, nucleic acid conformation and cell wall carbohydrates. Our results provide new interesting evidences that SPs specifically interact with PAHs in defining bacteria metabolic profiles and further emphasize the importance of studying the interaction of bacteria with their surrounding matrix to deeply understand PAHs degradation in soils.

Sub-micron level investigation reveals the inaccessibility of stabilized carbon in soil microaggregates

Direct evidence-based approaches are vital to evaluating newly proposed theories on the persistence of soil organic carbon and establishing the contributions of abiotic and biotic controls. Our primary goal was to directly identify the mechanisms of organic carbon stabilization in native-state, free soil microaggregates without disrupting the aggregate microstructure using scanning transmission x-ray microscopy coupled with near edge x-ray absorption fine structure spectroscopy (STXM-NEXAFS). The influence of soil management practices on microaggregate associated-carbon was also assessed. Free, stable soil microaggregates were collected from a tropical agro-ecosystem in Cruz Alta, Brazil. The long-term experimental plots (>25 years) comparing two tillage systems: no-till and till with a complex crop rotation. Based on simultaneously collected multi-elemental associations and speciation, STXM-NEXAFS successfully provided submicron level information on organo-mineral associations. Simple organic carbon sources were found preserved within microaggregates; some still possessing original morphology, suggesting that their stabilization was not entirely governed by the substrate chemistry. Bulk analysis showed higher and younger organic carbon in microaggregates from no-till systems than tilled systems. These results provide direct submicron level evidence that the surrounding environment is involved in stabilizing organic carbon, thus favoring newly proposed concepts on the persistence of soil organic carbon.

Fermentative Production of N-Methylglutamate From Glycerol by Recombinant Pseudomonas putida.

N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L−1 N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L−1.

CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coli.

Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.

Uniformly expanded interlayer distance to enhance the rate performance of soft carbon for lithium-ion batteries

Various carbons with novel structure attract tremendous interests as anode materials for high-rate batteries due to their rapid lithium-ion transfer; practically, they often deliver low initial cycle coulombic efficiency and serious decay for the large surface area. Here, we report a new soft carbon (SC) electrode prepared by using single and simple carbon sources. The carbon possesses a relatively uniform interlayer distance of 0.37 nm (equal to the interlayer distance of LiC6). Taken as a high-rate anode, SC delivers a remarkable discharge capacity of 169 mAh g−1 at a current density of 20 C (1 C = 372 mAh g−1) after 1000 cycles, which is tenfold higher compared to those of commercial graphitic carbon anodes. In the case of low surface area, the achievement is attributed to rapid ion transport and intercalation in proper interlayer distance causing slight change, which result in high-rate and stability property.