Discovery of HyT-Based Degraders of CDK9-Cyclin T1 Complex.

Direct modulation of the non-kinase functions of cyclin and CDK-cyclin complexes poses challenges. We utilize hydrophobic tag (HyT) based small-molecule degraders induced degradation of cyclin T1 and its corresponding kinase partner CDK9. LL-CDK9-12 demonstrated the most potent and selective degradation ability, with DC50 values of 0.362 µM against CDK9 and 0.680 µM against cyclin T1. In prostate cancer cells, LL-CDK9-12 showed enhanced anti-proliferative activity than its parental molecule SNS032 and LL-K9-3, the previous reported CDK9-cyclin T1 degrader. Moreover, LL-CDK9-12 suppressed the downstream signaling of CDK9 and AR efficiently. Altogether, LL-CDK9-12 was an effective dual degrader of CDK9-cyclin T1 and helped study the unknown function of CDK9-cyclin T1. These results suggest that HyT-based degraders could be used as a strategy to induce the degradation of protein complexes, providing insights for the design of protein complexes' degraders.

Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli.

γ-Aminobutyric acid (GABA) is used widely in various fields, such as agriculture, food, pharmaceuticals, and biobased chemicals. Based on glutamate decarboxylase (GadBM4) derived from our previous work, three mutants, GadM4-2, GadM4-8, and GadM4-31, were obtained by integrating enzyme evolution and high-throughput screening methods. The GABA productivity obtained through whole-cell bioconversion using recombinant Escherichia coli cells harboring mutant GadBM4-2 was enhanced by 20.27% compared to that of the original GadBM4. Further introduction of the central regulator GadE of the acid resistance system and the enzymes from the deoxyxylulose-5-phosphate-independent pyridoxal 5'-phosphate biosynthesis pathway resulted in a 24.92% improvement in GABA productivity, reaching 76.70 g/L/h without any cofactor addition with a greater than 99% conversion ratio. Finally, when one-step bioconversion was applied for the whole-cell catalysis in a 5 L bioreactor, the titer of GABA reached 307.5 ± 5.94 g/L with a productivity of 61.49 g/L/h by using crude l-glutamic acid (l-Glu) as the substrate. Thus, the biocatalyst constructed above combined with the whole-cell bioconversion method represents an effective approach for industrial GABA production.

Native Amino Group Directed Site-Selective ε-C(sp2)-H Iodination of Primary Amines.

Selective remote C-H activating amines using unmodified NH2 as a native directing group demonstrate compelling synthetic utilities. The 3-arylpropan-1-amine moiety is present in many drugs and candidates in clinical trials. Selective iodination of 3-arylpropan-1-amines on remote aryl rings gives valuable intermediates for modifying bioactive molecules and synthesizing quinolones. Here we report the first palladium-catalyzed selective ε-C(sp2)-H iodination of free 3-arylpropan-1-amines via a seven-membered palladacycle.

[Advances in physiological activities and synthesis of ß-nicotinamide mononucleotide].

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and ß isomer is its active form. Studies have shown that ß-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of ß-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize ß-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of ß-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of ß-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of ß-NMN.

Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol.

BACKGROUND: Pyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements. RESULTS: A high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room­temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter. CONCLUSION: The characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

[Advances on microbial synthesis of L-proline and trans-4-hydroxy-L-proline].

L-proline (L-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-L-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of L-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of "green, environmental protection and high efficiency". In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of L-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of L-Pro and T-4-Hyp. This paper reviews the application and production methods of L-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of L-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of L-Pro and T-4-Hyp, aiming to provide a theoretical basis for the "green bio-manufacturing" of L-Pro and T-4-Hyp and promote their industrial production.

Interaction of magnolia bark extracts with Staphylococcus aureus DNA and evaluation of the stability of their antibacterial activities.

Magnolia bark is an edible traditional Chinese medicine that has antibacterial activity against Staphylococcus aureus. In the present study, interactions between S. aureus DNA and raw magnolia bark (RMB) and ginger mix-fried magnolia bark (GMB) aqueous extracts were determined via spectroscopic methods. Fluorescence spectroscopy and Stern-Volmer constants showed that S. aureus DNA quenched the fluorescence of the extracts by static quenching. UV-Vis spectroscopy and iodide quenching experiments indicated that the interactions between S. aureus DNA and the fluorescent substances might involve groove binding or electrostatic interactions. In 4', 6-diamidino-2-phenylindole competitive assays, the fluorescence intensity at decreased as the extract amount was increased. This indicates that groove binding is responsible for the fluorescence quenching. The antibacterial activity of GMB aqueous extract treated under light, cold, heat and cycling hot-cold conditions decreased by 13.99, 9.31, 10.89 and 14.40%, respectively, whereas that of RMB aqueous extract treated under the same conditions decreased by 8.91, 14.99, 14.99 and 13.70%, respectively. The results indicate that S. aureus DNA quenches the fluorescence of GMB and RMB aqueous extracts by grooving interactions. Additionally, the antibacterial activities of GMB and RMB extracts are sensitive to light and temperature, respectively.

[New targeted compounds-biosynthesis of phytocannabinoids].

Phytocannabinoids are bioactive terpenoids that are exclusive to Cannabis sativa L. The main pharmacologically active phytocannabinoids are Δ9-tetrahydrocannabinol and cannabidiol, both target endogenous cannabinoid receptors. Δ9-tetrahydrocannabinol and cannabidiol have extensive therapeutic potential due to their participation in many physiological and pathological processes in human body by activating the endocannabinoid system. At present, Δ9-tetrahydrocannabinol, cannabidiol and their analogues or combination preparations are used to treat epilepsy, vomiting in patients with cancer chemotherapy, spasticity in multiple sclerosis and relieve neuropathic pain and pain in patients with advanced cancer. With the further exploration of the application value of Δ9-tetrahydrocannabinol and cannabidiol as well as the increasing demand for standardization of pharmaceutical preparations, it is imminent to achieve large-scale production of Δ9-tetrahydrocannabinol and cannabidiol in the pharmaceutical industry. In this article, pharmacological research progress of phytocannabinoids in recent years, biosynthetic pathways of phytocannabinoids and the mechanism of key enzymes as well as various product development strategies of cannabinoids in pharmaceutical industry are reviewed. By exploring the potential of synthetic biology as an alternative strategy for the source of phytocannabinoids, it will provide a theoretical basis for the research and development of microbial engineering for cannabinoids synthesis, and promote the large-scale production of medicinal cannabinoids.

Pyrroloquinoline quinone protects against exercise-induced fatigue and oxidative damage via improving mitochondrial function in mice.

Pyrroloquinoline quinone (PQQ) has a variety of biological functions. However, rare attention has been paid to its effects on exercise-induced damage. Here, we assessed the potential protective effects of PQQ against the fatigue and oxidative damage caused by repeated exhaustive exercise, and studied the underlying mechanism. The models for exercise-induced fatigue were established, and the parameters were measured, including the time to exhaustion (TTE), biochemical indicators, the expression of nuclear factor kappa B (NF-κB) and inflammatory cytokines and so on. Besides, the mitochondrial function was evaluated by the morphology, membrane potential, respiratory function, adenosine triphosphate (ATP) levels, and the application of the mitochondrial complex I inhibitor. The results demonstrate that PQQ prolongs TTE, causes the decrease in the activity of serum creatine kinase and lactate dehydrogenase, increases the activity of antioxidant enzymes, inhibits the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and diminishes the over expression of NF-κB (p65) and inflammatory mediators. Furthermore, PQQ preserves normal mitochondrial function. Particularly, PQQ reduces the accumulation of ROS triggered by the mitochondrial complex I inhibitor. These data suggest that PQQ can significantly protect mice from exercise-induced fatigue and oxidative damage by improving mitochondrial function. These data also suggest that PQQ controls mitochondrial activity through directly affecting the NADH dehydrogenase.

Two-stage oxygen supply strategy for enhancing fed-batch production of pyrroloquinoline quinone in Hyphomicrobium denitrificans FJNU-6.

Oxygen is a vital parameter for pyrroloquinoline quinone (PQQ) biosynthesis. In this study, the effects of oxygen supply on the biosynthesis of PQQ were first investigated systematically with Hyphomicrobium denitrificans FJNU-6. Following a kinetic analysis of the specific cell growth rate (µx) and specific PQQ formation rate (µp) in 5 L benchtop fermentation systems at various oxygen supply levels ranging from 0 to 60%, a novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity. Moreover, the transcription of genes involved in methanol oxidation and PQQ biosynthesis was analyzed throughout the process to outline the effect of oxygen supply on cell metabolism. Furthermore, with constant feeding of methanol at 0-1 g/L after the initial methanol was consumed completely, the PQQ concentration and productivity reached 1070 mg/L and 7.64 mg/L/h, respectively, after 140 h in a 5-L fermenter. The two-stage oxygen supply strategy developed in this study provides an effective and economical strategy for the industrial production of PQQ.Key Points• A novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity.•The transcription of genes involved in methanol oxidation and PQQ biosynthesis was regulated by changes in oxygen supply.• This study offers an effective and economical strategy for industrial or large-scale production of PQQ.

[Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication].

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.

Identification and characterization of a novel alkalistable and salt-tolerant esterase from the deep-sea hydrothermal vent of the East Pacific Rise.

A novel esterase gene selected from metagenomic sequences of deep-sea hydrothermal vents was successfully expressed in Escherichia coli. The recombinant protein (est-OKK), which belongs to the lipolytic enzyme family V, exhibited high activity toward pNP-esters with short acyl chains and especially p-nitrophenyl butyrate. Site-mutagenesis results confirmed that est-OKK contains the nonclassical catalytic tetrad predicted by alignment and computational modeling. The est-OKK protein is a moderately thermophilic enzyme that is relatively thermostable, and highly salt-tolerant, which remained stable in 3 mol/L NaCl for 6 hr. The est-OKK protein showed the considerable alkalistability, displayed optimal activity at pH 9.0 and maintained approximately 70% of its residual activity after incubation at pH 10 for 4 hr. Furthermore, the est-OKK activity was strongly resistant to a variety of metal ions such as Co2+ , Zn2+ , Fe2+ , Na+ , and K+ ; nonionic detergents such as Tween-20, Tween-80; and organic solvents such as acetone and isopropanol. Taken together, the novel esterase with unique characteristics may give us a new insight into the family V of lipolytic enzymes, and could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions or food and pharmaceutical industries.

Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst.

We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15 g/L GABA at 45 °C from L-glutamic acid (L-Glu) with a productivity of 40.94 g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65 g/L of GABA with 99.14 mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3 mol% conversion of 3 M L-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40 g/L of GABA and a 61.48 g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40 h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04 g/L/h productivity with a 99.6 mol% conversion of 3 M L-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed to date, even better than the productivity obtained from the three successive conversion cycles.

Whole-cell conversion of l-glutamic acid into gamma-aminobutyric acid by metabolically engineered Escherichia coli.

A simple and high efficient way for the synthesis of gamma-aminobutyric acid (GABA) was developed by using engineered Escherichia coli as whole-cell biocatalyst from l-glutamic acid (l-Glu). Codon optimization of Lactococcus lactis GadB showed the best performance on GABA production when middle copy-number plasmid was used as expression vector in E. coli BW25113. The highest production of GABA reached 308.96 g L(-1) with 99.9 mol% conversion within 12 h, when E. coli ΔgabAB (pRB-lgadB) concentrated to an OD600 of 15 in 3 M l-Glu at 45 °C. Furthermore, the strain could be reused at least three cycles in 2 M crude l-Glu with an average productivity of 40.94 g L(-1) h(-1). The total GABA yield reached 614.15 g L(-1) with a molar yield over 99 %, which represented the highest GABA production ever reported. The whole-cell bioconversion system allowed us to achieve a promising cost-effective resource for GABA in industrial application.

[Construction of Saccharomyces cerevisiae mutant with knockout of SNF4 gene].

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kan(r) as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kan(r) marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-deltaSNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach ofSNF4 gene knockout by Cre-LoxP system.

[Expression of a pectin lyase A gene from Aspergillus niger in Pichia pastoris GS115].

In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.