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At the 3' end of the C2 gene in the mammalian TRB locus, a distinct reverse TRBV30 gene (named TRBV31 in mice) has been conserved throughout evolution. In the fully annotated TRB locus of 14 mammals (including six orders), we observed noteworthy variations in the localization and quality of the reverse V30 genes and Recombination Signal Sequences (RSSs) in the gene trees of 13 mammals. Conversely, the forward V29 genes and RSSs were generally consistent with the species tree of their corresponding species. This finding suggested that the evolution of the reverse V30 gene was not synchronous and likely played a crucial role in regulating adaptive immune responses. To further investigate this possibility, we utilized single-cell TCR sequencing (scTCR-seq) and high-throughput sequencing (HTS) to analyze TCRβ CDR3 repertoires from both central and peripheral tissues of Primates (Homo sapiens and Macaca mulatta), Rodentia (Mus musculus: BALB/c, C57BL/6, and Kunming mice), Artiodactyla (Bos taurus and Bubalus bubalis), and Chiroptera (Rhinolophus affinis and Hipposideros armige). Our investigation revealed several novel observations: (1) The reverse V30 gene exhibits classical rearrangement patterns adhering to the '12/23 rule' and the 'D-J rearrangement preceding the V-(D-J) rearrangement'. This results in the formation of rearranged V30-D2J2, V30-D1J1, and V30-D1J2. However, we also identified 'special rearrangement patterns' wherein V30-D rearrangement preceding D-J rearrangement, giving rise to rearranged V30-D2-J1 and forward Vx-D2-J. (2) Compared to the 'deletional rearrangement' (looping out) of forward V1-V29 genes, the reverse V30 gene exhibits preferential utilization with 'inversional rearrangement'. This may be attributed to the shorter distance between the V30 gene and D gene and the 'inversional rearrangement' modes. In summary, in the mammalian TRB locus, the reverse V30 gene has been uniquely preserved throughout evolution and preferentially utilized in V(D)J recombination, potentially serving a significant role in adaptive immunity. These results will pave the way for novel and specialized research into the mechanisms, efficiency, and function of V(D)J recombination in mammals. [ABSTRACT FROM AUTHOR]

Background: 2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable pseudoaromatic dicarboxylic acid, represents a promising building block for the manufacture of biodegradable polyesters. Microbial production of PDC has been extensively investigated, but low titers and yields have limited industrial applications. Results: In this study, a multi-step biosynthesis strategy for the microbial production of PDC was demonstrated using engineered Escherichia coli whole-cell biocatalysts. The PDC biosynthetic pathway was first divided into three synthetic modules, namely the 3-dehydroshikimic acid (DHS) module, the protocatechuic acid (PCA) module and the PDC module. Several effective enzymes, including 3-dehydroshikimate dehydratase for the PCA module as well as protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase for the PDC module were isolated and characterized. Then, the highly efficient whole-cell bioconversion systems for producing PCA and PDC were constructed and optimized, respectively. Finally, the efficient multi-step biosynthesis of PDC from glucose was achieved by smoothly integrating the above three biosynthetic modules, resulting in a final titer of 49.18 g/L with an overall 27.2% molar yield, which represented the highest titer for PDC production from glucose reported to date. Conclusions: This study lays the foundation for the microbial production of PDC, including one-step de novo biosynthesis from glucose as well as the microbial transformation of monoaromatics. [ABSTRACT FROM AUTHOR]

Verticillium wilt in cotton caused by V erticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V . dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated B acillus amyloliquefaciens 41 B-1, exhibited greater than 50% biocontrol efficacy against V . dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41 B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V . dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V . dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V . dahliae. [ABSTRACT FROM AUTHOR]

Objective: The liver plays a dual role in regulating temperature and immune responses. Examining the influence of Heat stress (HS) on liver T cells contributes significantly to understanding the intricate interplay between the immune system and hepatic tissues under thermal stress. This study focused on investigating the characteristics of the T-cell receptor (TCR) β chain CDR3 repertoire in bovine liver samples under both HS and pair-fed (PF) environmental conditions., Methods: Sequencing data from six samples sourced from the GEO database underwent annotation. Utilizing immunarch and VDJtool software, the study conducted comprehensive analyses encompassing basic evaluation, clonality assessment, immune repertoire comparison, diversity estimation, gene usage profiling, VJ gene segment pairing scrutiny, clonal tracking, and Kmers analysis., Results: All four TCR chains, namely α, β, γ, and δ, were detected, with the α chains exhibiting the highest detection frequency, followed closely by the β chains. The prevalence of αβ TCRs in bovine liver samples underscored their crucial role in governing hepatic tissue's physiological functions. The TCR β CDR3 repertoire showcased substantial inter-individual variability, featuring diverse clonotypes exhibiting distinct amino acid lengths. Intriguingly, HS cattle displayed heightened diversity and clonality, suggesting potential peripheral T cell migration into the liver under environmental conditions. Notably, differential VJ gene pairings were observed in HS cattle compared to the PF, despite individual variations in V and J gene utilization. Additionally, while most high-frequency amino acid 5-mers remained consistent between the HS and PF, GELHF and YDYHF were notably prevalent in the HS group. Across all samples, a prevalent trend of high-frequency 5mers skewed towards polar and hydrophobic amino acids was evident., Conclusion: This study elucidates the characteristics of liver TCR β chain CDR3 repertoire under HS conditions, enhancing our understanding of HS implications.

In nature, chirality is a common phenomenon and closely related to life, also significantly influences the properties of the substance. The chemical synthesis of chiral pharmaceutical chemicals has encountered challenges such as poor atom economy and process economy, serious environmental pollution and waste of the resource. The biosynthesis route has the advantages of high selectivity and environmental-friendliness. In recent years, the rapid developments in the accessible key enzymes, understanding of catalytic mechanism, construction of new synthetic pathways of optical pure intermediates, process development and scale-up production have made it possible to address the challenges encountered in the production of active pharmaceutical ingredients, thus promoting a green and sustainable pharmaceutical industry in China. This review summarized the achievements made in this field by researchers at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.

Aromatic compounds make up a large part of fragrances and are traditionally produced by chemical synthesis and direct extraction from plants. Chemical synthesis depends on petroleum resources and has disadvantages such as causing environment pollutions and harsh reaction conditions. Due to the low content of aromatic compounds in plants and the low yield of direct extraction, plant extractions require large amounts of plant resources that occupy arable land. In recent years, with the development of metabolic engineering and synthetic biology, microbial synthesis of aromatic compounds from renewable resources has become a promising alternative approach to traditional methods. This review describes the research progress on the synthesis of aromatic fragrances by model microorganisms such as Escherichia coli or yeast, including the synthesis of vanillin through shikimic acid pathway and the synthesis of raspberry ketone through polyketide pathway. Moreover, this review highlights the elucidation of native biosynthesis pathways, the construction of synthetic pathways and metabolic regulation for the production of aromatic fragrances by microbial fermentation.

Metabolic engineering has been developed for nearly 30 years since the early 1990s, and it has given a great impetus to microbial strain breeding and improvement. Aromatic chemicals are a variety of important chemicals that can be produced by microbial fermentation and are widely used in the pharmaceutical, food, feed, and material industry. Microbial cells can be engineered to accumulate a variety of useful aromatic chemicals in a targeted manner through rational engineering of the biosynthetic pathways of shikimate and the derived aromatic amino acids. This review summarizes the metabolic engineering strategies and biosynthetic pathways for the production of aromatic chemicals developed in the past 30 years, with the aim to provide a valuable reference and promote the research in this field.

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or Kan(R) onto the chromosome of V. natriegens could reach 4×10⁻⁵ and 4×10⁻⁴, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.

Long noncoding RNAs (lncRNAs) are associated with tumor development and progression. LncRNA UCA1 (UCA1) recently has been reported to take part in cancer cell proliferation. However, the expression and underlying molecular mechanism of UCA1 in cervical cancer cell glycolysis is unclear. This study aimed to investigate the role of UCA1 in cervical cancer. In order to explore the role of UCA1 in cervical cancer, first, the expression levels of UCA1 in cervical cancer tissues were measured, and the results showed that UCA1 levels were higher in cancer tissues compared to matched adjacent normal tissues. The inhibition of UCA1 expression suppressed human cervical cancer cell proliferation and glycolysis. Additionally, our experimental results indicated that UCA1 could directly bind to miR-493-5p and regulate miR-493-5p expression in an inverse manner. Namely, UCA1 could reverse the inhibitory effect of miR-493-5p on cervical cancer cells' proliferation and glycolysis. Moreover, we revealed that HK2 is a target gene of miR-493-5p through a Targetscan prediction. It was verified that miR-493-5p downregulated HK2 mRNA and protein levels using real time RT-PCR and Western blotting. In a summary, this study demonstrated that UCA1 functioned as an oncogene by UCA1/miR-493-5p/HK2 axis in cervical cancer., Competing Interests: None., (IJCEP Copyright © 2018.)