Three dimensional conformations of transcription factor stabilized bacterial chromosomes to adapt to environmental stress.
■ Our reporter Li Chen
In hot weather, a cup of sweet fruit juice will soon go bad, and sometimes it will produce alcohol flavor. This is probably a "brewing" bacteria that is good at converting glucose and fructose into ethanol - zymomonas mobilis.
However, this "culprit" that can naturally produce ethanol is a potential stock in the eyes of scientists - qualified to become a chassis cell like yeast and Escherichia coli to manufacture bulk chemicals for humans.
He Mingxiong, a researcher from Chengdu Research Institute of Biogas Science (hereinafter referred to as the Institute of Biogas Science) of the Ministry of Agriculture and Rural Affairs, has analyzed the three-dimensional conformation of chromosomes of Zymomonas mobilis adapted to environmental stress, revealing the molecular mechanism of transcription factors widely existing in prokaryotes that mediate the three-dimensional conformation of chromosomes and regulate the expression of stress resistant genes to cope with environmental stress. Recently, the research paper was published online in Nucleic Acid Research.
The reviewers believe that this is a very interesting discovery - chemical molecules and transcription factors affect the three-dimensional conformation of bacterial chromosomes to promote or inhibit gene transcription. This research not only provides a new scientific basis for understanding the relationship between the structure and function of prokaryotic genomes, but also lays a foundation for rational design of engineering strains from the perspective of three-dimensional genomes.
Cell factory "potential stock": bacteria producing ethanol naturally
As a bacterium that can convert glucose and fructose into ethanol, Zymomonas mobilis has a special physiological and biochemical characteristic. He Mingxiong, the corresponding author of the paper, said that the reason why it can produce ethanol depends on the specific enzyme system in its metabolic pathway. These enzyme systems let it first convert carbon sources (such as glucose) into pyruvate, and then continue to convert them into ethanol and carbon dioxide.
If agricultural and forestry wastes such as straw and other biomass can be converted into glucose or fructose and then treated by this bacterium, wouldn't it be "convenient" to obtain industrial ethanol?
Moreover, studies have found that Zymomonas mobilis also shows broad application prospects in food, health, medicine and other fields.
However, biomass resources such as straw are dense, difficult to degrade and difficult to use. In the process of transformation, pretreatment is needed to release glucose and other nutrients. However, the hydrolysate produced by pretreatment has many microbial fermentation inhibitors, such as acetic acid, furan formaldehyde, phenols and salt compounds. Among them, acetic acid and furaldehyde are two main toxic by-products.
"These inhibitors will significantly reduce the transformation efficiency of microbial fermentation." He Mingxiong said that if strains resistant to inhibitors can be screened, it is expected to find relevant resistance genes.
So, seven years ago, Wang Weiting, the master student of He Mingxiong's team, set out to do strain mutation screening, with the goal of screening out stress resistant strains that can tolerate these inhibitors, that is, strains that can efficiently use sugars to produce target products under stress to resist interference from these inhibitors.
Through genome recombination and other technologies, Wang Weiting finally bred ZM532, a strain of ZM532 with stress resistance characteristics, which greatly improved its biotransformation efficiency.
After obtaining resistant strains, the research team wants to further understand why these strains produce stress resistance. They used genome re sequencing, transcriptomics, proteomics and other common analysis methods, but the analysis results were unexpected - although some of the genes were related to the stress resistant phenotype, they still could not fully explain the mechanism of significant changes in the stress resistant phenotype.
Traditional means "fail" and embark on the journey of exploring the three-dimensional structure of genome
"Our team used genome re sequencing technology and transcriptomics analysis to reveal the mechanism of stress resistance in the early stage." Chen Mao, the first author of the paper and a doctor from Biogas Research Institute, said that it was found that mutations such as single nucleotide mutation (SNP), fragment insertion and deletion occurred in the genome of the stress resistant strain of Zymomonas mobilis.
They also tried to find the cause of stress resistance phenotype from genes associated with genome mutations, but most of these genes associated with mutations are unrelated to stress resistance.
In 2019, Samina Shabbir, a doctoral student, began to do transcriptome and proteome analysis, trying to discover key regulatory factors and explain the mechanism of stress resistance at the transcriptional level. However, the results could not explain the reasons for the dramatic changes in phenotype.
"These results show that there is a mismatch between genome mutation and stress resistance phenotype in Zymomonas mobilis." He Mingxiong said that genome mutation is a common phenomenon in bacteria to adapt to stress environment. "People often think that these mutations will lead to changes in bacterial stress resistance phenotype, but sometimes on the one-dimensional level, that is, at the gene level, it may not be possible to explain why this phenotype changes."
Although it has been known that in eukaryotes, genomic mutations may change the three-dimensional structure of chromosomes, leading to cancer and other diseases, little is known about the relationship between genomic mutations and chromosome conformation in prokaryotes.
Chromosome conformation is the organization and arrangement of chromosomes in three-dimensional space, including chromosome folding, interaction between chromosome regions, etc. These higher-order structures have important effects on gene expression and regulation.
Inspired by the relevant research on three-dimensional genomics of eukaryotes, He Mingxiong's team proposed the hypothesis: whether it is genome mutations that lead to changes in the three-dimensional structure of chromosomes, thus leading to the occurrence of stress resistant phenotypes. Therefore, traditional methods such as genomics, transcriptomics, proteomics and other one-dimensional and two-dimensional technologies are difficult to find the mechanism behind stress resistant phenotypes.
In 2020, Chen Mao, who is pursuing a doctoral degree, embarked on a journey of exploring the three-dimensional genome structure, trying to verify the function of key regulatory factors, analyze the dynamic changes of the three-dimensional genome, and reveal the molecular mechanism of stress resistance regulation.
Accelerate the breeding process of "cell factory"
How do genomic mutations and environmental stresses (such as acetic acid and furaldehyde) affect the three-dimensional chromosome conformation of Zymomonas mobilis?
Wu Bo, co first author of the paper and associate researcher of Biogas Research Institute, introduced that in 3D genome research, interactions between sites hundreds of kb apart on the DNA one-dimensional level are usually called long-range interactions, while interactions tens of kb or even smaller are called short-range interactions.
"Our research found that genome mutations only changed the local short-range interaction; when acetic acid and furaldehyde were stressed, not only the long-range interaction but also the short-range interaction was changed. These short-range interactions constituted the chromosome domain, which is the basic structural unit of the three-dimensional conformation of chromosomes." Wu Bo said.
They further analyzed the boundary characteristics of the domain and found an important class of transcription factors, namely the iron absorption regulatory protein (Fur) family. In Zymomonas mobilis, the regulatory protein family also includes zinc absorption regulatory protein (Zur). "If these two related genes are knocked out, it will seriously affect the strain's resistance to reverse acetic acid and furaldehyde phenotype," said Chen Mao.
He Mingxiong explained that bacterial transcription factors are crucial for bacteria to adapt to adversity, because these proteins can regulate a series of gene expression, thus affecting the physiological metabolism of bacteria. Previous studies mainly focused on the most important function of transcription factors - regulating gene expression, and expounded the importance of these regulations from a one-dimensional level. However, transcription factors may affect three-dimensional conformation while regulating gene expression on chromosomes.
"Our research not only shows that transcription factors have the ability to regulate gene expression to adapt to the environment, but also maintain the stability of the three-dimensional conformation of chromosomes." He Mingxiong said that this is the first time that global transcription factors have the ability to regulate the three-dimensional conformation of chromosomes in bacteria.
This research explains the core molecular biology problem of "structure determines function" from a new perspective, reveals the molecular mechanism of stress resistant phenotype formation, and provides a new scientific basis for understanding the relationship between structure and function of prokaryotic genomes.
He Mingxiong emphasized that the analysis of the molecular mechanism of stress resistance is conducive to the rational design of strains directly. The so-called rational design is based on a deep understanding of microbiology, metabolic pathways and genomics to achieve specific breeding goals through precise design and regulation of microbial genomes, metabolic pathways or physiological characteristics. Focusing on the influence of chromosome interaction on rational design from the perspective of chromosome structure can improve the efficiency of phenotypic evolution.
"Our research is still in the initial stage, and we will actively explore the application of three-dimensional genome in synthetic biology in the future." He Mingxiong said that understanding the stress resistance mechanism of zymomonas mobilis is expected to efficiently use agricultural and forestry wastes such as straw, and even develop a new "cell factory".
Relevant paper information:
https://doi.org/10.1093/nar/gkae318
