Intrinsically unidirectional chemically fuelled rotary molecular motors.

Biological systems mainly utilize chemical energy to fuel autonomous molecular motors, enabling the system to be driven out of equilibrium1. Taking inspiration from rotary motors such as the bacterial flagellar motor2 and adenosine triphosphate synthase3, and building on the success of light-powered unidirectional rotary molecular motors4-6, scientists have pursued the design of synthetic molecular motors solely driven by chemical energy7-13. However, designing artificial rotary molecular motors operating autonomously using a chemical fuel and simultaneously featuring the intrinsic structural design elements to allow full 360° unidirectional rotary motion like adenosine triphosphate synthase remains challenging. Here we show that a homochiral biaryl Motor-3, with three distinct stereochemical elements, is a rotary motor that undergoes repetitive and unidirectional 360° rotation of the two aryl groups around a single-bond axle driven by a chemical fuel. It undergoes sequential ester cyclization, helix inversion and ring opening, and up to 99% unidirectionality is realized over the autonomous rotary cycle. The molecular rotary motor can be operated in two modes: synchronized motion with pulses of a chemical fuel and acid-base oscillations; and autonomous motion in the presence of a chemical fuel under slightly basic aqueous conditions. This rotary motor design with intrinsic control over the direction of rotation, simple chemical fuelling for autonomous motion and near-perfect unidirectionality illustrates the potential for future generations of multicomponent machines to perform mechanical functions.

Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid.

Insufficient biosynthesis efficiency during the lipogenic phase can be a major obstacle to engineering oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs). Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in an engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases (KCSs) rationally guided reconstructing an efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (as the major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts.

Engineering Escherichia coli to produce aromatic chemicals from ethylene glycol.

Microbial overproduction of aromatic chemicals has gained considerable industrial interest and various metabolic engineering approaches have been employed in recent years to address the associated challenges. So far, most studies have used sugars (mostly glucose) or glycerol as the primary carbon source. In this study, we used ethylene glycol (EG) as the main carbon substrate. EG could be obtained from the degradation of plastic and cellulosic wastes. As a proof of concept, Escherichia coli was engineered to transform EG into L-tyrosine, a valuable aromatic amino acid. Under the best fermentation condition, the strain produced 2 g/L L-tyrosine from 10 g/L EG, outperforming glucose (the most common sugar feedstock) in the same experimental conditions. To prove the concept that EG can be converted into different aromatic chemicals, E. coli was further engineered with a similar approach to synthesize other valuable aromatic chemicals, L-phenylalanine and p-coumaric acid. Finally, waste polyethylene terephthalate (PET) bottles were degraded using acid hydrolysis and the resulting monomer EG was transformed into L-tyrosine using the engineered E. coli, yielding a comparable titer to that obtained using commercial EG. The strains developed in this study should be valuable to the community for producing valuable aromatics from EG.

The Effect of Intramolecular Hydrogen Bonds on the Rotational Barriers of the Biaryl C-C Axis.

Axially chiral compounds are attracting more attention recently. Although hydrogen bonds are reported as a vital weak force that influences the properties of compounds, the effect of intramolecular hydrogen bonds on the atropisomerization of the Caryl -Caryl single bonds has not yet been well quantitatively investigated. Here, a series of axially chiral biaryl compounds were synthesized to study the effect of hydrogen bonds on the rotational barriers of the biaryl C-C axis. Experimental studies demonstrated that the rotational barrier of hydrogen bonding biaryl 9 was significantly lower (46.7 kJ mol-1 ) than biaryl 10 without hydrogen bonds. Furthermore, theoretical studies revealed that the intramolecular hydrogen bond stabilized the transition state (TS) of tri-ortho-substituted biaryl 9, relieving the steric repulsion in the TS. We believe that this study will provide chemists with a deeper understanding of the atropisomerization process of axially chiral biaryl compounds.

Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli.

Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.

Upcycling chitin-containing waste into organonitrogen chemicals via an integrated process.

Chitin is the most abundant renewable nitrogenous material on earth and is accessible to humans in the form of crustacean shell waste. Such waste has been severely underutilized, resulting in both resource wastage and disposal issues. Upcycling chitin-containing waste into value-added products is an attractive solution. However, the direct conversion of crustacean shell waste-derived chitin into a wide spectrum of nitrogen-containing chemicals (NCCs) is challenging via conventional catalytic processes. To address this challenge, in this study, we developed an integrated biorefinery process to upgrade shell waste-derived chitin into two aromatic NCCs that currently cannot be synthesized from chitin via any chemical process (tyrosine and l-DOPA). The process involves a pretreatment of chitin-containing shell waste and an enzymatic/fermentative bioprocess using metabolically engineered Escherichia coli The pretreatment step achieved an almost 100% recovery and partial depolymerization of chitin from shrimp shell waste (SSW), thereby offering water-soluble chitin hydrolysates for the downstream microbial process under mild conditions. The engineered E. coli strains produced 0.91 g/L tyrosine or 0.41 g/L l-DOPA from 22.5 g/L unpurified SSW-derived chitin hydrolysates, demonstrating the feasibility of upcycling renewable chitin-containing waste into value-added NCCs via this integrated biorefinery, which bypassed the Haber-Bosch process in providing a nitrogen source.

Synthesis of Oxygenated Sesquiterpenoids Enabled by Combining Metabolic Engineering and Visible-Light Photocatalysis.

The diversification of natural products to expand biologically relevant chemical space for drug discovery can be achieved by combining complementary bioprocessing and chemical transformations. Herein, genetically engineered Escherichia coli fermentation to produce amorphadiene and valencene was combined with metal-free photocatalysis transformations to further access nootkatone, cis-nootkatol and two hydration derivatives. In fermentation, using a closed, anaerobic condition avoided the use of organic overlay, increased the productivity, and simplified the work-up process. Metal-free photocatalysis hydration and allylic C-H oxidation were designed and implemented to make the whole process greener. It was shown that the anti-Markovnikov selectivity of photocatalyzed alkene hydration could be reversed by stereo-electronic and steric effects existing in complex natural products. The combination of bioprocessing and photocatalysis may provide an efficient and greener way to expand the chemical space for pharmaceutical, flavor and fragrance industry.

para-Selective Radical Trifluoromethylation of Benzamide Derivatives via Iminium Intermediates.

The direct C-H trifluoromethylation of arenes via a radical pathway has attracted considerable attention recently. However, a major challenge of C-H trifluoromethylation is the lack of site-selectivity on the phenyl ring especially para-selectivity. Herein we show a new strategy for para-selective C-H trifluoromethylation of benzamide derivatives using iminium activation. The reaction undergoes a radical-type nucleophilic substitution instead of a radical-type electrophilic substitution owing to iminium activation as a result of lowering the LUMO (lowest unoccupied molecular orbital). A wide range of substrates are compatible with this method giving almost exclusive para-trifluoromethylated products.

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds.

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 h when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 h. We also engineered a E. coli strain to produce 24 mg/L of prenol in an ethanol-containing medium, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.

A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases.

Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat /km value toward (S)-2-octanol compared to its wild type.

Monoterpenoid biosynthesis by engineered microbes.

Monoterpenoids are C10 isoprenoids and constitute a large family of natural products. They have been used as ingredients in food, cosmetics, and therapeutic products. Many monoterpenoids such as linalool, geraniol, limonene, and pinene are volatile and can be found in plant essential oils. Conventionally, these bioactive compounds are obtained from plant extracts by using organic solvents or by distillation method, which are costly and laborious if high-purity product is desired. In recent years, microbial biosynthesis has emerged as alternative source of monoterpenoids with great promise for meeting the increasing global demand for these compounds. However, current methods of production are not yet at levels required for commercialization. Production efficiency of monoterpenoids in microbial hosts is often restricted by high volatility of the monoterpenoids, a lack of enzymatic activity and selectivity, and/or product cytotoxicity to the microbial hosts. In this review, we summarize advances in microbial production of monoterpenoids over the past 3 years with particular focus on the key metabolic engineering strategies for different monoterpenoid products. We also provide our perspective on the promise of future endeavors to improve monoterpenoid productivity.

Using biopolymer bodies for encapsulation of hydrophobic products in bacterium.

Producing some small hydrophobic molecules in microbes is challenging. Often these molecules cannot cross membranes, and thus their production may be limited by lack of storage space in the producing organism. This study reports a new technology for in vivo storage of valuable hydrophobic products in/on biopolymer bodies in Escherichia coli. A biodegradable and biocompatible polyester - poly (3-hydroxybutyrate) (PHB) - was selected as the intracellular storage vessel to encapsulate lycopene, which is a chromogenic model compound. The hydrophobic interaction between lycopene and PHB was verified by using in vitro binding test and sucrose density gradient centrifugation. Further in vivo characterization was performed by using Confocal Laser Scanning Microscopy (CLSM). The images validated the in vivo co-localization between PHB granules and lycopene. The images also showed that lycopene aggregated in bacteria that did not produce PHB, which may challenge the commonly accepted hypothesis that most lycopene molecules are stored in cell membranes of recombinant host. We also confirmed that producing PHB did not negatively affect lycopene biosynthesis in the E. coli strains and collected data suggesting that PHB titer and lycopene titer were positively correlated when the cells were engineered to co-produce them. The biopolymers that encapsulated hydrophobic molecules could have many useful applications, especially in controlled release because the polymers are biodegradable, and the encapsulated products would be released during the polymer degradation.

N-(2-Aminoethyl)-2-(hexylthio) Acetamide-Functionalized Pillar[5]arene for the Selective Detection of l-Trp through Guest-Adaptive Multisupramolecular Interactions.

Tryptophan (Trp) is very necessary for biosystems; therefore, high-efficient detection of Trp is an important subject. Hereof, based on our early research works on fluorescent sensors, we rationally designed and synthesized a fluorescent sensor (SNP5) based on N-(2-aminoethyl)-2-(hexylthio) acetamide-functionalized pillar[5]arene, which showed high selectivity and sensitive recognition for l-Trp (LOD = 2.19 × 10-8 M). Moreover, SNP5 exhibited aggregation-induced emission enhancement fluorescence. Within SNP5, the pillar[5]arene group could act as N-H···π- and C-H···π-interaction sites, as well as a H-bond-interaction site; meanwhile, the N-(2-aminoethyl)-2-(hexylthio) acetamide group also served as a multihydrogen-bonding site. As a result, SNP5 could selectively detect l-Trp through the synergy of the pillar[5]arene group and the N-(2-aminoethyl)-2-(hexylthio) acetamide group. Compared with previous work, the results of this work support the strategy that changing the functionalized group of the pillar[5]arene can adjust the selectivity of the pillar[5]arene-based sensor and achieve the detection of different amino acids. The detection mechanism was specifically researched through experiments and theoretical calculations including frontier orbitals, electrostatic potential, and the independent gradient model approach. Interestingly, these theoretical calculations not only supported the experimental results but also provided a visualized understanding of guest-adaptive multisupramolecular interactions between SNP5 and l-Trp.

An azine-containing bispillar[5]arene-based multi-stimuli responsive supramolecular pseudopolyrotaxane gel for effective adsorption of rhodamine B.

An azine-containing bispillar[5]arene was designed and synthesized by the reaction of aldehyde functionalized-pillar[5]arene and hydrazine. Then, a novel bispillar[5]arene-based supramolecular pseudopolyrotaxane has been successfully prepared via host-guest interaction. Interestingly, by taking advantage of the host-guest interactions, π-π stacking interactions and hydrogen bonding interactions, the multi-stimuli-responsive gel-sol phase transitions of such a supramolecular pseudopolyrotaxane gel were successfully realized under different stimuli, such as acid, temperature, concentration, and competitive guests. Moreover, this supramolecular system could effectively adsorb dye molecule rhodamine B. It is worth noting that this supramolecular pseudopolyrotaxane gel prepared in cyclohexanol solution (BP5·G·C) could be used as an adsorbent material for adsorbing rhodamine B with adsorption efficiency of 98.4%. Meanwhile, the adsorption efficiency was 97.6% for supramolecular pseudopolyrotaxane gel prepared in DMSO-H2O (v : v, 8 : 2) binary solution (BP5·G·D), also indicating the superior adsorption effect of BP5·G·D toward the dye molecule rhodamine B.

Aggregation-induced emission supramolecular organic framework (AIE SOF) gels constructed from tri-pillar[5]arene-based foldamer for ultrasensitive detection and separation of multi-analytes.

In this study, a novel aggregation-induced emission supramolecular organic framework (AIE SOF) with ultrasensitive response, termed FSOF, was constructed using a tri-pillar[5]arene-based foldamer. Interestingly, benefiting from the noise signal shielding properties of FSOF as well as the competition between the cationπ and ππ interactions, the FSOF shows an ultrasensitive response for multi-analytes, such as Fe3+, Hg2+ and Cr3+. The limits of detection (LODs) of the FSOF for Fe3+, Hg2+ and Cr3+ are in the range of 9.40 × 10-10-1.86 × 10-9. More importantly, the xerogel of FSOF exhibits porous mesh structures, which could effect high-efficiency separation above metal ions from their aqueous solution, with adsorption percentages in the range 92.39-99.99%. In addition, by introducing metal ions into the FSOF, a series of metal ions coordinated supramolecular organic frameworks (MSOFs) were successfully constructed. Moreover, MSOFs show selective fluorescence "turn on" ultrasensitive detection CN- (LOD = 2.12 × 10-9) and H2PO4- (LOD = 1.78 × 10-9). This is a novel approach to construct SOFs through a tri-pillar[5]arene-based foldamer, and also provides a new way to achieve ultrasensitive detection and high-efficiency separation.

Short, auto-inducible promoters for well-controlled protein expression in Escherichia coli.

Expression of recombinant proteins in Escherichia coli often requires use of inducible promoters to shorten the lag phase and improve protein productivity and final protein titer. Synthetic molecules that cannot be metabolized by E. coli, such as isopropyl thiogalactopyranoside (IPTG), have been frequently used to trigger the protein expression during early exponential growth phase. This practice has many drawbacks, including high cost and toxicity of IPTG, complex operating procedure, and non-uniform protein expression pattern (some cells in the population do not express recombinant proteins). A few auto-inducible protein expression systems have been developed recently to overcome some of these limitations, but they required use of an additional plasmid or presence of large (a few kilobases) DNA part to be functional, making plasmid construction to be difficult, especially when multiple genes need to be expressed. In this study, by using RNA sequencing, we identified a short, endogenous promoter (PthrC) that can be auto-induced during early exponential growth phase, and improved its performance by use of native and mutated regulatory elements. We found that the developed mutants of PthrC drove uniform protein expression-close to 100% of cells were fluorescent when green fluorescence protein was used as target protein-and cells carrying them could achieve much higher cell density than those with T7 promoter (PT7), a commonly used inducible promoter. In terms of promoter strength (product protein quantity per cell), the developed promoter mutants can cover a range of strength, from 30 to 150% of maximal strength of PT7. One strong mutant (PthrC3_8) was found to work well at a large range of temperature (22, 30, 37 °C) and in various media, and was also confirmed to cause less stress to host cell than PT7 when they were used to express a toxic protein. We foresee that PthrC3 and its mutants will be useful genetic parts for various applications including metabolic engineering and biocatalysis.

High-level production of Arthrobacter aurescens CYC705 nitrilase in Escherichia coli for biosynthesis of iminodiacetic acid.

Nitrilase from Arthrobacter aurescens CYC705 can hydrolyze the iminodiacetonitrile to iminodiacetic acid (IDA) efficiently, and its high-level production in Escherichia coli has not been established. In the present work, the production of this nitrilase expressed in E. coli BL21(DE3) with a recombinant plasmid pET28a-cyc705 was optimized. Various culture conditions and process parameters including medium components and concentrations, inducer types and concentrations, inducing temperature and time were systematically examined in a shake flask. After optimization, the OD600 , nitrilase activity, and productivity were obviously improved and achieved to 40.91 ± 1.341, 98.12 ± 1.248 U/mL, and 2,230 ± 28.36 U L(-1)  H(-1) , respectively, about 2.1-, 30-, and 33-fold increases as compared with those in the primary medium. Furthermore, four different fermentation strategies were adopted to scale up cultivation of the recombinant E. coli BL21(DE3)/pET28a-cyc705 in a 3.7-L fermenter. Substituting the peanut powder with fish peptone and accompanying with 1.0% glycerol feeding could significantly reduce the bubble production and shorten the fermentation time, which resulted in a nitrilase productivity of 4,653 ± 38.16 U L(-1) H(-1) that was about two times higher than that in a shake flask. The high-level production of A. aurescens CYC705 nitrilase established in this study will meet the need of industrial biosynthesis of IDA.

High-level soluble and functional expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli.

D-Amino acid oxidase is an important biocatalyst used in a variety of fields, and its economically justified level recombinant expression in Escherichia coli has not been established. To accomplish this, after a single Phe54Tyr substitution, fusion proteins of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) with 6 × His-tags were constructed and expressed in E. coli. The effects of his-tags fusing position were revealed. Significant increase in holoenzyme percent and protein solubility made N-terminus tagged TvDAO (termed NHDAO) a suitable choice for TvDAO production. However, reduced cell growth and protein production rates were also observed for the NHDAO bearing strains. To optimize the performance of NHDAO production, changes of culture medium were tested. Finally, a production of 140 U/mL or 3.48 g active enzyme per liter which accounted for 41.4 % of the total protein, and a specific activity of 16.68 U/mg for the crude extract, were achieved in a 3.7 L fermenter in 28.5 h. This indicated a possibility for functional and economical TvDAO expression in E. coli to meet the industrial need.

Fabrication and characterization of anthocyanin-loaded double Pickering emulsions stabilized by ß-cyclodextrin.

Anthocyanins, one of the important water-soluble pigments, are sensitive to environmental factors, which limits the application of anthocyanins in food field. In order to overcome this limitation, double Pickering emulsions stabilized by ß-cyclodextrin were developed. The optimum preparation conditions of the emulsions were determined firstly and the performance and structure of emulsions were investigated. Results showed that the optimum preparation conditions of emulsions were the ratio of (W1/O): W2 = 6:4 and 4 % ß-cyclodextrin concentration. Optical microscope and confocal laser scanning microscope results confirmed that ß-cyclodextrin adsorbed onto the surface of droplets forming stable double Pickering emulsions structure. In vitro gastrointestinal digestion experiments proved that double Pickering emulsions played a controlled-release effect in the small intestine. Rheological analysis proved that the emulsions exhibited elastic properties and demonstrated shear thinning behavior. The emulsions showed excellent stability under centrifugation and thermal conditions. These findings will promote anthocyanins' application in daily diet.