Design and Evolution of an Enzyme for the Asymmetric Michael Addition of Cyclic Ketones to Nitroolefins by Enamine Catalysis.

Consistent introduction of novel enzymes is required for developing efficient biocatalysts for challenging biotransformations. Absorbing catalytic modes from organocatalysis may be fruitful for designing new-to-nature enzymes with novel functions. Herein we report a newly designed artificial enzyme harboring a catalytic pyrrolidine residue that catalyzes the asymmetric Michael addition of cyclic ketones to nitroolefins through enamine activation with high efficiency. Diverse chiral γ-nitro cyclic ketones with two stereocenters were efficiently prepared with excellent stereoselectivity (up to 97% e.e., >20:1 d.r.) and good yield (up to 86%). This work provides an efficient biocatalytic strategy for cyclic ketone functionalization and highlights the usefulness of artificial enzymes for extending biocatalysis to further non-natural reactions.

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution.

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

Effect of ultrasonic modification on the binding ability of pectin to anthocyanin.

BACKGROUND: Pectin was considered as a potential candidate to improve the thermal stability of anthocyanins, and the binding ability of pectin to anthocyanins was influenced by its structure. In this study, sunflower pectins, modified by ultrasound (40 kHz) for different periods of time, were prepared and used to bind with anthocyanins, extracted from purple sweet potato. RESULTS: Characterization and thermal stability of pectin-anthocyanin complexes were investigated. The ultrasonic modification of pectin resulted in many changes in pectin chemical structure, including degradation of neutral sugar side chains, breakage of methoxyl groups, and increased molecular flexibility. Extension of ultrasonic modification time led to greater changes in pectin chemical structure. Analysis of the binding ability, as determined by Fourier transform infrared spectroscopy and molecular dynamics simulations, revealed that the interaction between pectin and anthocyanins was driven by hydrogen bonding, electrostatic interaction, and hydrophobic interaction. Pectins with different ultrasonic modification times bound with anthocyanins to different extents, mainly resulting from an increase in the number of hydrogen bonds. According to high-performance liquid chromatographic analysis, during heating at 90 °C the stronger the binding ability of pectin and anthocyanin complex, the better was its thermal stability. CONCLUSION: Ultrasonic modification of pectin could effectively enhance its binding ability to anthocyanin. © 2023 Society of Chemical Industry.

Effects of sunflower pectin on thermal stability of purple sweet potato anthocyanins at different pH.

The present study aimed to examine the impact of sunflower pectin (SFP) on the thermal stability and antioxidant activity of purple sweet potato anthocyanins (PSPA) at varying pH levels. It was observed that the pH value significantly influenced the ability of pectin to protect anthocyanins from thermal degradation, which was found to be associated with the rate of binding between PSPA and SFP. The binding rate of PSPA-SFP was observed to be highest at pH 4.0, primarily due to the influence of electrostatic interaction and hydrogen bonding. Monoacylated anthocyanins exhibited a binding rate approximately 2-4 % higher than that of diacylated anthocyanins. The PSPA-SFP demonstrated its highest thermal stability at pH 4.0, with a corresponding half-life of 14.80 h at 100 °C. Molecular dynamics simulations indicated that pectin had a greater affinity for the flavylium cation and hemiketal form of anthocyanins. The antioxidant activity of anthocyanins in PSPA and PSPA-SFP increased with increasing pH, suggesting that anthocyanins at high pH had higher antioxidant activity than anthocyanins at low pH.

Effects of different re-fermentation methods on the quality characteristics of kombucha beverages.

The effects of different re-fermentation methods on the quality characteristics of kombucha beverages were investigated. The quality characteristics of kombucha beverages included the basic physicochemical indicators (pH, total acidity, reducing sugar, total sugar, organic acids, total phenolic compound, total flavonoid compound), antioxidant activity, volatile flavor substance and sensory evaluation of the beverages. The results showed the re-fermentation methods including the mixed fermentation and the step-by-step fermentation significantly decreased total acidity and various organic acids (P < 0.05) than traditional kombucha with no re-fermentation. In addition, the contents of total phenol compounds and total flavonoid compounds for the step-by-step fermentation were 184.70 and 338.33 mg/L respectively, and were higher compared with mixed fermentation and traditional kombucha with no re-fermentation. The antioxidant activity in the step-by-step fermentation was much stronger than that of mixed fermentation and traditional kombucha with no re-fermentation. Moreover, there were 53 kinds of volatile flavor compounds produced in the step-by-step fermentation, 14 of them were unique with good sensory quality. In conclusion, the re-fermentation methods for traditional kombucha (the step-by-step fermentation and mixed fermentation) had more active ingredients and better sensory quality, and the step-by-step fermentation was better than mixed fermentation.

Improving (2S)-naringenin production by exploring native precursor pathways and screening higher-active chalcone synthases from plants rich in flavonoids.

Flavonoids are a group of valuable compounds with a variety of health benefits. (2 S)-Naringenin is an important flavonoid skeleton, which can be tailored into almost all flavonoids. In this study, the Saccharomyces cerevisiae native precursor pathways were explored and higher-active CHSs from plants rich in flavonoids were screened. The results indicated that overexpressing the native precursor pathways is not an efficient approach to improving (2 S)-naringenin production in our chassis strain. On the other hand, by screening from plants rich in flavonoids, we obtained four CHSs with higher activities than the commonly used PhCHS. Among these CHSs, SjCHS1 increased the (2 S)-naringenin titer by 48.38% in shaking flasks. Finally, we combined the native precursor pathways optimization with the higher-active CHS that screened, and further increased the (2 S)-naringenin titer to 203.49 mg/L from glucose in shaking flasks. The results achieved in this study indicated that plants rich in flavonoids are good sources for higher-active CHS screening, and that the heterologous pathway and chassis precursor flux should be synergistically engineered to achieve higher production.

Optimum chalcone synthase for flavonoid biosynthesis in microorganisms.

Chalcones and the subsequently generated flavonoids, as well as flavonoid derivatives, have been proven to have a variety of physiological activities and are widely used in: the pharmaceutical, food, feed, and cosmetic industries. As the content of chalcones and downstream products in native plants is low, the production of these compounds by microorganisms has gained the attention of many researchers and has a history of more than 20 years. The mining and engineering of chalcone synthase (CHS) could be one of the most important ways to achieve more efficient production of chalcones and downstream products in microorganisms. CHS has a broad spectrum of substrates, and its enzyme activity and expression level can significantly affect the efficiency of the biosynthesis of flavonoids. This review summarizes the recent advances in the: structure, mechanism, evolution, substrate spectrum, transformation, and expression regulation in the flavonoid biosynthesis of this vital enzyme. Future development directions were also suggested. The findings may further promote the research and development of flavonoids and health products, making them vital in the fields of human diet and health.

A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica.

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.

Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production.

Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.

Genome Sequence of Klebsiella pneumoniae CICC10011, a Promising Strain for High 2,3-Butanediol Production.

Klebsiella pneumoniae CICC10011, a promising 2,3-butanediol producer, has received much attention because of its high productivity. Here, the first draft genome sequence of this efficient strain may provide the genetic basis for further insights into the metabolic and regulatory mechanisms underlying the production of 2,3-butanediol at a high titer.

Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R)-2,3-butanediol.

Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.

Genome Sequence of Paenibacillus polymyxa ATCC 12321, a Promising Strain for Optically Active (R,R)-2,3-Butanediol Production.

Paenibacillus polymyxa is a potential strain for (R,R)-2,3-butanediol production. Here, we report an annotated draft genome sequence of P. polymyxa strain ATCC 12321, which contains 4,429 protein-coding genes and 49 structural RNAs. This genome sequence provides a genetic basis for a better understanding of the mechanism for the accumulation of highly optically active (R,R)-2,3-butanediol.