Deep learning offers new approaches to investigate the mechanisms underlying complex biological phenomena, such as subgenome dominance. Subgenome dominance refers to the dominant expression and/or biased fractionation of genes in one subgenome of allopolyploids, which has shaped the evolution of a large group of plants. However, the underlying cause of subgenome dominance remains elusive. Here, we adopt deep learning to construct two convolutional neural network (CNN) models, binary expression model (BEM) and homoeolog contrast model (HCM), to investigate the mechanism underlying subgenome dominance using DNA sequence and methylation sites. We apply these CNN models to analyze three representative polyploidization systems, Brassica, Gossypium, and Cucurbitaceae, each with available ancient and neo/synthetic polyploidized genomes. The BEM shows that DNA sequence of the promoter region can accurately predict whether a gene is expressed or not. More importantly, the HCM shows that the DNA sequence of the promoter region predicts dominant expression status between homoeologous gene pairs retained from ancient polyploidizations, thus predicting subgenome dominance associated with these events. However, HCM fails to predict gene expression dominance between new homoeologous gene pairs arising from the neo/synthetic polyploidizations. These results are consistent across the three plant polyploidization systems, indicating broad applicability of our models. Furthermore, the two models based on methylation sites produce similar results. These results show that subgenome dominance is associated with long-term sequence differentiation between the promoters of homoeologs, suggesting that subgenome expression dominance precedes and is the driving force or even the determining factor for sequence divergence between subgenomes following polyploidization.

BACKGROUND: P. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methanol or other inducible promoters, natural or engineered, have defects in either fermentation safety or expression capacity. The utilization of chemical inducers typically adds complexity to the product purification process, but there is no other well-controlled protein synthesis system than promoters yet.
OBJECTIVE: The study aimed to address the aforementioned challenges by constructing light-regulated gene transcription and protein translation systems with excellent expression capacity and light sensitivity.
METHODS: Trans-acting factors were designed by linking the N. crassa blue-light sensor WC-1 with the activation domain of endogenous transcription factors. Light inducible or repressive promoters were then constructed through chimeric design of cis-elements (light-responsive elements, LREs) and endogenous promoters. Various configurations of trans-acting factor/LRE pairs, along with different LRE positions and copy numbers were tested for optimal promoter performance. In addition to transcription, a light-repressive translation system was constructed through the \"rare codon brake\" design. Rare codons were deliberately utilized to serve as brakes during protein synthesis, which were switched on and off through the light-regulated changes in the expression of the corresponding pLRE-tRNA.
RESULTS: As demonstrated with GFP, the light-inducible promoter 4pLRE-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP in darkness. As for the light-repressive translation system, the \"triple brake\" design successfully eliminated leakage and achieved light repression on protein synthesis without any impact on mRNA expression.
CONCLUSIONS: The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.

5-Hydroxytryptophan (5-HTP), a precursor of the neurotransmitter serotonin in mammals, has demonstrated efficacy in treating various diseases such as depression, fibromyalgia and obesity. However, conventional biosynthesis methods of 5-HTP are limited by low yield and high reagent and process costs. In this study, the strain C1T7-S337A/F318Y with optimized promoter distribution was obtained, and the 5-HTP yield was 60.30 % higher than that of the initial strain. An efficient fermentation process for 5-HTP synthesis was developed using strain C1T7-S337A/F318Y with whey powder as a substrate for cell growth and inducer production. Shake flask fermentation experiments yielded 1.302 g/L 5-HTP from 2.0 g/L L-tryptophan (L-Trp), surpassing the whole-cell biocatalysis by 42.86 %. Scale-up to a 5 L fermenter further increased the yield to 1.649 g/L. This fermentation strategy substantially slashed reagent cost by 95.39 %, providing a more economically viable and environmentally sustainable route for industrial biosynthesis of 5-HTP. Moreover, it contributes to the broader utilization of whey powder in various industries.

Promoters are DNA sequences that bind with RNA polymerase to initiate transcription, regulating this process through interactions with transcription factors. Accurate identification of promoters is crucial for understanding gene expression regulation mechanisms and developing therapeutic approaches for various diseases. However, experimental techniques for promoter identification are often expensive, time-consuming, and inefficient, necessitating the development of accurate and efficient computational models for this task. Enhancing the model\'s ability to recognize promoters across multiple species and improving its interpretability pose significant challenges. In this study, we introduce a novel interpretable model based on graph neural networks, named GraphPro, for multi-species promoter identification. Initially, we encode the sequences using k-tuple nucleotide frequency pattern, dinucleotide physicochemical properties, and dna2vec. Subsequently, we construct two feature extraction modules based on convolutional neural networks and graph neural networks. These modules aim to extract specific motifs from the promoters, learn their dependencies, and capture the underlying structural features of the promoters, providing a more comprehensive representation. Finally, a fully connected neural network predicts whether the input sequence is a promoter. We conducted extensive experiments on promoter datasets from eight species, including Human, Mouse, and Escherichia coli. The experimental results show that the average Sn, Sp, Acc and MCC values of GraphPro are 0.9123, 0.9482, 0.8840 and 0.7984, respectively. Compared with previous promoter identification methods, GraphPro not only achieves better recognition accuracy on multiple species, but also outperforms all previous methods in cross-species prediction ability. Furthermore, by visualizing GraphPro\'s decision process and analyzing the sequences matching the transcription factor binding motifs captured by the model, we validate its significant advantages in biological interpretability. The source code for GraphPro is available at https://github.com/liuliwei1980/GraphPro.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

The main components of sandalwood heartwood essential oil are terpenoids, approximately 80% of which are α-santalol and β-santalol. In the synthesis of the main secondary metabolites of sandalwood heartwood, the key gene, santalene synthase (SaSSY), can produce α-santalene and β-santalene by catalyzed (E, E)-FPP. Furthermore, santalene is catalyzed by the cytochrome monooxygenase SaCYP736A167 to form sandalwood essential oil, which then produces a fragrance. However, the upstream regulatory mechanism of the key gene santalene synthase remains unclear. In this study, SaSSY (Sal3G10690) promoter transcription factors and SaSSY cis-elements were screened. The results showed that the titer of the sandalwood cDNA library was 1.75 × 107 CFU/mL, 80% of the inserted fragments identified by PCR were over 750 bp in length, and the positivity rate of the library was greater than 90%. The promoter region of the SaSSY gene was shown to have the structural basis for potential regulatory factor binding. After sequencing and bioinformatics analysis, we successfully obtained 51 positive clones and identified four potential SaSSY transcriptional regulators. Sal6G03620 was annotated as the transcription factor MYB36-like, and Sal8G07920 was annotated as the small heat shock protein HSP20 in sandalwood. Sal1G00910 was annotated as a hypothetical protein of sandalwood. Sal4G10880 was annotated as a homeobox-leucine zipper protein (ATHB-15) in sandalwood. In this study, a cDNA library of sandalwood was successfully constructed using a yeast one-hybrid technique, and the transcription factors that might interact with SaSSY gene promoters were screened. This study provides a foundation for exploring the molecular regulatory mechanism involved in the formation of sandalwood heartwood.

As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

Traditionally, immunoglobulin (Ig) expression has been attributed solely to B cells/plasma cells with well-documented and accepted regulatory mechanisms governing Ig expression in B cells. Ig transcription is tightly controlled by a series of transcription factors. However, increasing evidence has recently demonstrated that Ig is not only produced by B cell lineages but also by various types of non-B cells (non-B-Ig). Under physiological conditions, non-B-Ig not only exhibits antibody activity but also regulates cellular biological activities (such as promoting cell proliferation, adhesion, and cytoskeleton protein activity). In pathological conditions, non-B-Ig is implicated in the development of various diseases including tumour, kidney disease, and other immune-related disorders. The mechanisms underline Ig gene rearrangement and transcriptional regulation of Ig genes in non-B cells are not fully understood. However, existing evidence suggests that these mechanisms in non-B cells differ from those in B cells. For instance, non-B-Ig gene rearrangement occurs in an RAG-independent manner; and Oct-1 and Oct-4, rather than Oct-2, are required for the transcriptional regulation of non-B derived Igs. In this chapter, we will describe and compare the mechanisms of gene rearrangement and expression regulation between B-Ig and non-B-Ig.

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the -555 to -1 region. The prediction of the transcription start site of TNFSF11 gene-2000 ∼ + 500bp showed that there was a CpG site in 17 ∼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b, Myog, Trl, and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene.