Butanol Tolerance of Lactiplantibacillus plantarum: A Transcriptome Study

Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

Clostridium acetobutylicum bacterial membrane responses to carbon ion beam irradiation perturbations

Background Clostridium acetobutylicum is an important strain during acetone-butanol-ethanol (ABE) fermentation. However, butanol has toxic effects on cells, limiting the application of ABE fermentation. Accordingly, in this study, we aimed to elucidate the metabolic mechanisms through which Clostridium adapts to butanol stress to facilitate the industrial utilization of Clostridium. Results First, using cell morphology, cell membrane permeability and membrane potential, cell surface hydrophobicity, and cell membrane fatty acid composition analyses in wild-type (ATCC 824) and butanol-tolerant (Y217) strains under butanol stress, we explored the responses in the cell membrane to evaluate the damage caused by butanol poisoning. After 2.0% (v/v) butanol treatment, the extracellular conductivity of ATCC 824 increased, intracellular proteins and nucleotides were released in large quantities, the fluorescein diacetate staining rate decreased, the membrane potential decreased, and the cell membrane permeability increased. Under butanol shock, the cell surface of Y217 cells remained intact, and its butanol tolerance mechanism increased the integrity of cell membrane and reduced leakage of cell contents caused by changed in cell membrane permeability, thereby preventing butanol damage to the cell membrane. When stimulated with butanol, Y217 cells showed reduced surface hydrophobicity, thereby improving cellular tolerance to butanol. A comparison of differences in fatty acid compositions between ATCC 824 and Y217 cell membranes under butanol stress further demonstrated that maintenance of the normal physiological characteristics of cell membranes played important roles in resisting the impact of organic solvents. Conclusions Our findings clarified the changes in physiological and biochemical characteristics of the mutant Y217 cell membrane stimulated with butanol to enhance its tolerance. These results may provide important theoretical guidance for further accelerating the acquisition of bacteria with high butanol tolerance and promoting butanol production. Moreover, our study provided a scientific basis for improving the industrial and environmental adaptability of Clostridium.

Tolerance against butanol stress by disrupting succinylglutamate desuccinylase inEscherichia coli

The four-carbon alcohol, butanol, is emerging as a promising biofuel and efforts have been undertaken to improve several microbial hosts for its production.

Meta-Analysis and Functional Validation of Nutritional Requirements of Solventogenic Clostridia Growing under Butanol Stress Conditions and Coutilization of d-Glucose and d-Xylose

ABSTRACTRecent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strainClostridium beijerinckiiSA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique.C. beijerinckiiSA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization ofd-glucose andd-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated thatC. beijerinckiiSA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli

ABSTRACT n-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including Escherichia coli, have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicates that n-butanol stress has components common to other stress responses, including perturbation of respiratory functions (nuo and cyo operons), oxidative stress (sodA, sodC, and yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, cpxR, and cpxP), and metabolite transport and biosynthesis (malE and opp operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during n-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during n-butanol stress were examined for increased sensitivity to n-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer, n-butanol-producing host.