Luis Caspeta

The heat shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in Escherichia coli . However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered E. coli strain (JU15), designed for d -lactic acid production, to enable cellular growth and fermentation of glucose at 45 °C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45 °C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved a controlled stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures. Key points: • The temperature upshift response of a thermally adapted strain in continuous culture was studied for the first time. • Genomic analyses revealed the presence of a double point mutation in the spoT gene. • The thermally adapted strain maintained underexpression of the spoT gene at high temperatures. • Supplementation of 0.15 g/L of hydrolyzed protein favored thermal adaptation at 45 °C.

Xylose is the most abundant pentose in nature. However, it is usually obtained in mixtures with glucose, leading to carbon catabolite repression in many microorganisms. Among E. coli lineages, significant metabolic and regulatory differences exist, requiring distinct metabolic engineering strategies to develop a xylose-selective phenotype in the strains W, K-12, and C. In this study, strain ES02 was engineered from Escherichia coli BL21 (DE3) as a xylose-selective strain by deleting the glk , ptsG , and manZ genes . However, when grown in a mixture of xylose and glucose, this strain’s specific growth rate and xylose consumption rate decreased by about 50% compared to cultures with only xylose. A modified version of the xylose-responsive transcriptional activator XylR ^Q31K was utilized to overcome this issue. The resulting strain ES04 (BL21 (DE3) Δglk , ΔmanZ , ΔptsG , xylR::Km ^r , lacZ::xylR ^C91A -Gm ^r ) efficiently used xylose as carbon source either alone or in a mixture with glucose, with a specific xylose consumption rate 75% higher than that of the wild-type strain BL21(DE3). Unexpectedly, strain ES04 partially recovers the ability to grow and consume glucose at a low rate, preferentially consuming xylose over glucose in sugar mixtures, revealing an altered carbon catabolite repression phenotype. Transcriptomics analysis suggested that glucose assimilation in this strain was related to the overexpression of the galactitol operon gatDCBAZY. Further inactivation of this operon confirmed its participation in glucose assimilation. Key points: • XylR ^Q31K alleviates carbon catabolite repression in the xylose-selective strain ES04. • Galactitol operon overexpression in ES04 links to partial glucose utilization. • ES04 strain preferentially uses xylose over glucose, revealing altered CCR.

Background: Escherichia coli JU15 is a metabolically engineered strain capable to metabolize C5 and C6 sugars with a high yield of d-lactic acid production at its optimal growth temperature (37 °C). The simultaneous saccharification and fermentation process allow to use lignocellulosic biomass as a cost-effective and high-yield strategy. However, this process requires microorganisms capable of growth at a temperature close to 50 °C, at which the activity of cellulolytic enzymes works efficiently. Results: The thermotolerant strain GT48 was generated by adaptive laboratory evolution in batch and chemostat cultures under temperature increments until 48 °C. The strain GT48 was able to grow and ferment glucose to d-lactate at 47 °C. It was found that a pH of 6.3 conciliated with GT48 growth and cellulase activity of a commercial cocktail. Hence, this pH was used for the SSF of a diluted acid-pretreated corn stover (DAPCS) at a solid load of 15% (w/w), 15 FPU/g-_DAPCS, and 47 °C. Under such conditions, the strain GT48 exhibited remarkable performance, producing d-lactate at a level of 1.41, 1.42, and 1.48-fold higher in titer, productivity, and yield, respectively, compared to parental strain at 45 °C. Conclusions: In general, our results show for the first time that a thermal-adapted strain of E. coli is capable of being used in the simultaneous saccharification and fermentation process without pre-saccharification stage at high temperatures.

Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological and whole-genome analyses were conducted to obtain insights on molecular responses which potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated by adaptive laboratory evolution (ALE) experiments. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H⁺, iron, and glycerol transport (i.e., PMA1, FRE1/2, JEN1, VMA2, VCX1, KHA1, AQY3, and ATO2); transcriptional regulation of stress responses to drugs, reactive oxygen species and heat-shock (i.e., HSF1, SKN7, BAS1, HFI1, and WAR1); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1, GPA1/2, RAS2, IRA2, and REG1). At 30 °C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results revealed that evolved strains adjust their intracellular pH by H⁺ and acetic acid transport, modify their metabolism and stress responses via glucose signaling pathways, control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides, and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Moreover, the motifs analysis in mutated transcription factors suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. Key points: • All the evolved strains overexpressed the plasma membrane H⁺-ATPase PMA1 at optimal conditions • Tolerant strain TAT12 mutated genes encoding weak acid and heat response TFs HSF1, SKN7, and WAR1 • TFs HSF1 and SKN7 likely controlled the transcription of metabolic genes associated to heat and acid tolerance

Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological, genomic, and transcriptomic analyses were conducted to obtain insights on molecular responses which could potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H ⁺ , iron, and glycerol transport (i.e. FRE1/2 , JEN1 , VMA2 , VCX1 , KHA1 , AQY3 , and ATO2 ); transcriptional regulation of stress responses to drugs, ROS and heat-shock (i.e. HSF1 , SKN7 , BAS1 , HFI1 , and WAR1 ); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1 , GPA1/2 , RAS2 , IRA2 and REG1 ). At 30°C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results from genomics and transcriptomics revealed that, as part of their adaptive responses, evolved yeast strains aimed to adjust their intracellular pH by H ⁺ and acetic acid transport; modify their metabolism and stress responses via glucose signaling pathways; control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides; and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Furthermore, tolerant strains did not show growth trade off under optimal ancestral conditions. Moreover, the motifs analysis in mutated TFs suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. This study provides insights into the molecular elements associated with yeast tolerance to acidic pH, acetic acid, and supraoptimal temperatures; information that can be used in inverse metabolic engineering.

Saccharomyces cerevisiae scarcely grows on minimal media with acetic acid, acidic pH, and high temperatures. In this study, the adaptive laboratory evoluti

Visualization is a key aspect of the analysis of omics data. Although many tools can generate pathway maps for visualization of yeast metabolism, they fail in reconstructing genome‐scale metabolic diagrams of compartmentalized metabolism. Here we report on the yeastGemMap, a process diagram of whole yeast metabolism created to assist data analysis in systems‐metabolic studies. The map was manually reconstructed with reactions from a compartmentalized genome‐scale metabolic model, based on biochemical process diagrams typically found in educational and specialized literature. The yeastGemMap consists of 3815 reactions representing 1150 genes, 2742 metabolites, and 14 compartments. Computational functions for adapting the graphical representation of the map are also reported. These functions modify the visual representation of the map to assist in three systems‐metabolic tasks: illustrating reaction networks, interpreting metabolic flux data, and visualizing omics data. The versatility of the yeastGemMap and algorithms to assist visualization of systems‐metabolic data was demonstrated in various tasks, including for single lethal reaction evaluation, flux balance analysis, and transcriptomic data analysis. For instance, visual interpretation of metabolic transcriptomes of thermally evolved and parental yeast strains allowed to demonstrate that evolved strains activate a preadaptation response at 30°C, which enabled thermotolerance. A quick interpretation of systems‐metabolic data is promoted with yeastGemMap visualizations.

Thermal damage, high osmolarity, and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentation to produce ethanol. We show that long‐term adaptive laboratory evolution at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated‐osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance. This maladaptation was bolstered by reestablishing proton homeostasis through increasing fermentation pH from 5 to 6 and/or adding potassium to the media. This change allowed the TTY23 strain to produce 1.3–1.6 times more ethanol than the parental strain in fermentations at 40°C with glucose concentrations ~300 g/L. Furthermore, ethanol titers and productivities up to 93.1 and 3.87 g·L ⁻¹·hr ⁻¹ were obtained from fermentations with 200 g/L glucose in potassium‐containing media at 40°C. Albeit the complexity of cellular responses to heat, ethanol, and high osmolarity, in this study we overcome such limitations by an inverse metabolic engineering approach.