Search Results (11)

Background: Dynamic changes in histone acetylation play crucial roles during cellular differentiation and disease development, but their detection in living cells is still a challenging task. Objectives: Here, we developed a Bimolecular Anchor Detector (BiAD) sensor for the detection of locus-specific changes in histone acetylation in living cells by fluorescence microscopy. Methods: We used the BRD9 bromodomain cloned as tandem double domain (2xBRD9-BD) as a reader of histone acetylation. It was integrated into a dual-color BiAD chassis that was previously described by us. Results: We identified the gene body of TTC34 as a potential target for our sensor, because it contains dense histone acetylation and 392 local sequence repeats. Using a binding-deficient mutant of 2xBRD9-BD as a negative control, we established a successful readout of histone acetylation at the TTC34 locus. A single-domain reader did not function, indicating the requirement for the double reader to enhance the affinity and specificity of the chromatin interaction via avidity effects. With this sensor, we could detect dynamic increases in histone acetylation at the TTC34 locus after the treatment of cells with the histone deacetylase inhibitor Trichostatin A for 6 h indicating the applicability of the sensor for single-cell epigenome studies. Conclusions: Our data demonstrate that active chromatin modifications can be detected by BiAD sensors using 2xBRD9-BD as a reader. This complements the toolkit of the available BiAD sensors and documents the modularity of BiAD sensors. Full article

The regulation of intracellular NADPH levels is currently a hotspot for research into bacterial modification and fermentation process optimization, and Corynebacterium glutamicum, an important industrial microorganism, achieves enhanced L-lysine production by regulating intracellular NADPH levels. In previous studies, transcriptome analysis was performed on C. glutamicum with different intracellular NADPH levels. The results showed that the expression level of transcription factor AtrN changed significantly. Moreover, experiments showed that transcription factor AtrN can sense high intracellular levels of NADPH and negatively regulate its synthesis. In this study, we integrated the pntAB gene of Escherichia coli into the genome of C. glutamicum XQ-5, successfully constructing a chassis cell with a high intracellular NADPH level. It was named TQ-1. On this basis, we knocked out and complemented the AtrN in strain TQ-1, resulting in strains TQ-2 and TQ-3, respectively. Then, the changes in cell growth, intracellular redox substances and cell membrane among these three strains were investigated. We found that the growth of TQ-2 was inhibited in the early growth stage and the cell survival rate was decreased because of the high increase in the intracellular NADPH level. In addition, the deletion of the AtrN gene also led to a decrease in the fluidity and an increase in the permeability of the cell membrane. Compared with TQ-1, TQ-3 showed slow growth only in the late growth stage, and the fluidity of its cell membrane was also enhanced. This indicates that AtrN guides the cells to make some adaptive changes to maintain cell growth when facing excessive intracellular reductive stress. This will facilitate future research on how potential upstream regulatory genes regulate AtrN and how AtrN regulates downstream genes to cope with cellular reductive stress. It also provides theoretical guidance for the specific modification of high-yield lysine-producing strains. Full article

Climate change and the scarcity of primary resources are driving the development of new, more renewable and environmentally friendly industrial processes. As part of this green chemistry approach, extremozymes (extreme microbial enzymes) can be used to replace all or part of the chemical synthesis stages of traditional industrial processes. At present, the production of these enzymes is limited by the cellular chassis available. The production of a large number of extremozymes requires extremophilic cellular chassis, which are not available. This is particularly true of halophilic extremozymes. The aim of this review is to present the current potential and challenges associated with the development of a haloarchaea-based cellular chassis. By overcoming the major obstacle of the limited number of genetic tools, it will be possible to propose a robust cellular chassis for the production of functional halophilic enzymes that can participate in the industrial transition of many sectors. Full article

Nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD⁺), is involved in the regulation of many physiological and metabolic reactions in the body. NMN can indirectly affect cellular metabolic pathways, DNA repair, and senescence, while also being essential for maintaining tissues and dynamic metabolic equilibria, promoting healthy aging. Therefore, NMN has found many applications in the food, pharmaceutical, and cosmetics industries. At present, NMN synthesis strategies mainly include chemical synthesis and biosynthesis. Despite its potential benefits, the commercial production of NMN by organic chemistry approaches faces environmental and safety problems. With the rapid development of synthetic biology, it has become possible to construct microbial cell factories to produce NMN in a cost-effective way. In this review, we summarize the chemical and biosynthetic strategies of NMN, offering an overview of the recent research progress on host selection, chassis cell optimization, mining of key enzymes, metabolic engineering, and adaptive fermentation strategies. In addition, we also review the advances in the role of NMN in aging, metabolic diseases, and neural function. This review provides comprehensive technical guidance for the efficient biosynthesis of NMN as well as a theoretical basis for its application in the fields of food, medicine, and cosmetics. Full article

1,4-diaminobutane is widely used in the industrial production of polymers, pharmaceuticals, agrochemicals and surfactants. Owing to economic and environmental concerns, there has been a growing interest in using microbes to produce 1,4-diaminobutane. However, there is lack of research on the influence of cofactors pyridoxal phosphate (PLP) and NADPH on the synthesis of 1,4-diaminobutane. PLP serves as a cofactor of ornithine decarboxylase in the synthesis of 1,4-diaminobutane. Additionally, the synthesis of 1 mol 1,4-diaminobutane requires 2 mol NADPH, thus necessitating consideration of NADPH balance in the efficient synthesis of 1,4-diaminobutane by Escherichia coli. The aim of this study was to enhance the synthesis efficiency of 1,4-diaminobutane through increasing production of PLP and NADPH. By optimizing the expression of the genes associated with synthesis of PLP and NADPH in E. coli, cellular PLP and NADPH levels increased, and the yield of 1,4-diaminobutane also increased accordingly. Ultimately, using glucose as the primary carbon source, the yield of 1,4-diaminobutane in the recombinant strain NAP19 reached 272 mg/L·DCW, by increased 79% compared with its chassis strain. Full article

Tausonia pullulans 6A7 is a low-temperature yeast strain that can produce lipases. Yeast, which is made up of chassis cells, is an important part of synthetic biology, and the use of the lipase-producing properties of T. pullulans 6A7 for the production of fatty acids provides a new pathway for targeted synthesis in yeast cell factories. In this study, we performed RNA-seq on lipase-producing T. pullulans 6A7 at different temperatures (15 °C, 20 °C, 20 °C without corn oil, and 25 °C). Therefore, a total of 8455 differentially expressed genes were screened, and 16 of them were FAD candidate genes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of group A (15 °C) vs. group D (25 °C) showed that the pathways of fatty acid biosynthesis (map00061) and the biosynthesis of unsaturated fatty acids (map01040) were significantly enriched. In the proposed temporal analysis of differentially expressed genes among the four temperature modulations, we found differentially expressed genes in nine clusters that had the same expression trends; these genes may be jointly involved in multiple biological processes in T. pullulans 6A7. In addition, we found 16 FAD candidate genes involved in fatty acid biosynthesis, and the expression of these genes had similar expression in the transcriptome trends with the different temperature treatments. These findings will help in future in-depth studies of the function and molecular mechanisms of these important FAD genes involved in fatty acid metabolism in yeast, and they could also be conducive to the establishment of a cellular factory for targeted fatty acid production by using yeast. Full article

Wax esters are widely distributed among microbes, plants, and mammals, and they serve protective and energy storage functions. Three classes of enzymes catalyze the reaction between a fatty acyl alcohol and a fatty acyl-CoA, generating wax esters. Multiple isozymes of two of these enzyme classes, the membrane-bound O-acyltransferase class of wax synthase (WS) and the bifunctional wax synthase/diacylglycerol acyl transferase (WSD), co-exist in plants. Although WSD enzymes are known to produce the wax esters of the plant cuticle, the functionality of plant WS enzymes is less well characterized. In this study, we investigated the phylogenetic relationships among the 12 WS and 11 WSD isozymes that occur in Arabidopsis, and established two in vivo heterologous expression systems, in the yeast Saccharomyces cerevisiae and in Arabidopsis seeds to investigate the catalytic abilities of the WS enzymes. These two refactored wax assembly chassis were used to demonstrate that WS isozymes show distinct differences in the types of esters that can be assembled. We also determined the cellular and subcellular localization of two Arabidopsis WS isozymes. Additionally, using publicly available Arabidopsis transcriptomics data, we identified the co-expression modules of the 12 Arabidopsis WS coding genes. Collectively, these analyses suggest that WS genes may function in cuticle assembly and in supporting novel photosynthetic function(s). Full article

The highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255-15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255-15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255-15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance. Full article

Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines. Full article

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression. Full article

The quest for a minimal cell not only sheds light on the fundamental principles of life but also brings great advances in related applied fields such as general biotechnology. Minimal cell projects came from the study of a plausible route to the origin of life. Later on, research extended and also referred to the construction of artificial cells, or even more broadly, as in vitro synthetic biology. The cell-free protein synthesis (CFPS) techniques harness the central cellular activity of transcription/translation in an open environment, providing the framework for multiple cellular processes assembling. Therefore, CFPS systems have become the first choice in the construction of the minimal cell. In this review, we focus on the recent advances in the quantitative analysis of CFPS and on its advantage for addressing the bottom-up assembly of a minimal cell and illustrate the importance of systemic chassis behavior, such as stochasticity under a compartmentalized micro-environment. Full article