Comprehensive network of stress-induced responses in Zymomonas mobilis during bioethanol production: from physiological and molecular responses to the effects of system metabolic engineering

Abstract

Nowadays, biofuels, especially bioethanol, are becoming increasingly popular as an alternative to fossil fuels. Zymomonas mobilis is a desirable species for bioethanol production due to its unique characteristics, such as low biomass production and high-rate glucose metabolism. However, several factors can interfere with the fermentation process and hinder microbial activity, including lignocellulosic hydrolysate inhibitors, high temperatures, an osmotic environment, and high ethanol concentration. Overcoming these limitations is critical for effective bioethanol production. In this review, the stress response mechanisms of Z. mobilis are discussed in comparison to other ethanol-producing microbes. The mechanism of stress response is divided into physiological (changes in growth, metabolism, intracellular components, and cell membrane structures) and molecular (up and down-regulation of specific genes and elements of the regulatory system and their role in expression of specific proteins and control of metabolic fluxes) changes. Systemic metabolic engineering approaches, such as gene manipulation, overexpression, and silencing, are successful methods for building new metabolic pathways. Therefore, this review discusses systems metabolic engineering in conjunction with systems biology and synthetic biology as an important method for developing new strains with an effective response mechanism to fermentation stresses during bioethanol production. Overall, understanding the stress response mechanisms of Z. mobilis can lead to more efficient and effective bioethanol production.

Keywords: Zymomonas mobilis; Bioethanol fermentation stress condition; Metabolic engineering; Stress response regulatory network; Synthetic biology; Systems biology.

Fig. 1

Bioethanol fermentation stress conditions and adaptation. Schematic view of process-related stress (osmotic, high temperature, lignocellulosic inhibitors, nutrient shortage, contamination with other microorganisms) and cellular-related stress (ethanol and oxidative stress) situations during bioethanol production that leads to activation of stress response mechanism in Z. mobilis

Fig. 2

Comparing compatible solute production mechanism inZ. mobilis and S. cerevisiae during high glucose and osmotic stress conditions. In Z. mobilis (a), expression of gfo gene encodes glucose-fructose oxidoreductase, and in S. cerevisiae (b), gpd1 gene encodes glycerol-3-phosphate dehydrogenase contributes to tolerance to high glucose concentrations

Fig. 3

Physiological and molecular stress responses inZ. mobilis during ethanol and lignocellulosic inhibitor stress conditions. a) Ethanol stress: Ethanol stress induces changes in the lipid membrane composition of Z. mobilis strains, resulting in a decrease in the level of unsaturated fatty acids to saturated fatty acids. During normal conditions, PspF binds to PspA protein. However, during ethanol stress, this protein is separated, leading to increased expression of the psp operon and high levels of PspA production. PspA proteins attach to the cell membrane structure, maintaining membrane structure and stability. Furthermore, during ethanol stress, the GFOR enzyme converts glucose to gluconolactate and sorbitol. The accumulation of sorbitol, as well as Hsp proteins and GroEL/GroES chaperones, controls protein conformation and folding. Interestingly, increasing the expression of tryptophan operon increases the level of this amino acid in the cytoplasm, preventing protein accumulation inside the cell. Additionally, the activity of regulatory RNAs (Zms), transcription factors such as TetR and sigma-70 enhance the expression of membrane transporters (ABC transporters and Ton-B), which significantly control membrane permeability under ethanol stress b) Lignocellulosic inhibitor stress: Under stress conditions, reactive oxygen species (ROS) such as OH⁻, O²⁻, and H₂O₂ may be produced, which can denature DNA structure. In response, DNA repair systems and proteins such as RecJ, DnaA, RecF, and UvrB are activated to maintain DNA conformation and stability. Regulatory networks such as LacI and sigma-70 increase the expression of genes that regulate TCA enzymes, leading to increased NADH and NADPH levels. Together with oxidoreductase enzymes, these cofactors convert lignocellulosic inhibitors such as furfural, HMF, and vanillin into low-toxic structures. The activity of Lon and DnaJ proteins helps retain protein structure and fold. Transcription factors such as TetR increase the expression of transmembrane proteins such as ABC transporters and RND, which inhibit the negative effects of lignocellulosic inhibitors and direct them outside the cell

Fig. 4

Physiological and molecular stress responses inZ. mobilis during high temperature and high glucose stress conditions. a) High-temperature stress: In Z. mobilis, high temperatures can negatively affect its cell performanceThe elevated temperature can disrupt DNA structure by breaking hydrogen bonds between nucleobases, resulting in denaturation. To counteract this, the DNA repair system is activated, aided by proteins such as RadA, RadC, and Xse, to stabilize and refold the DNA. Conversely, FtsH is inhibited during this stress condition. This protein typically binds to Sigma-32 to inhibit its activity and expression under normal conditions. However, in high-temperature stress conditions, FtsH inhibition activates Sigma-32, which, in conjunction with other chaperones and proteins, refolds denatured proteins and stabilizes their structure. Notably, WrbA protein binds to TrpR (the trp operon inhibitor) to increase tryptophan expression, preventing protein aggregation within the cytoplasm. In the periplasmic region, DegP and M16 proteins degrade misfolded proteins to prevent aggregation within the cell. Finally, increased expression of transmembrane proteins and MinC and MinD proteins helps regulate membrane structure and cell division under high-temperature stress, respectively b) High glucose stress: Elevating the concentration of glucose in the extracellular environment induces a series of intracellular signals that ultimately upregulate the expression of genes, such as the gfo gene. The increased activity of the GFOR enzyme facilitates the conversion of extracellular glucose into sorbitol. The accumulation of sorbitol inside the cell, in conjunction with chaperones like DnaJ, DnaK, and Hsp20, helps regulate and refold denatured proteins. Moreover, sorbitol enhances the expression of PDC and ADH enzymes, which control bioethanol production. Additionally, the transcription factors Sigma-54 and PspF increase the expression of the psp operon, promoting membrane stabilization during glucose stress. Transmembrane proteins, including TolC, play a crucial role in hemostasis by facilitating the flow of proteins and sugars outside the cell

Fig. 5

Systems metabolic engineering. Schematic view of systems metabolic engineering and its combination with classical metabolic engineering methods, systems biology and computational modeling, synthetic biology, and Adaptive laboratory evolution