Thermo-adaptive evolution of Corynebacterium glutamicum reveals the regulatory functions of fasR and hrcA in heat tolerance

Abstract

Background

High-temperature fermentation technology is promising in improving fermentation speed and product quality, and thereby widely used in various fields such as food, pharmaceuticals, and biofuels. However, extreme temperature conditions can disrupt cell membrane structures and interfere with the functionality of biological macromolecules (e.g. proteins and RNA), exerting detrimental effects on cellular viability and fermentation capability.

Results

Herein, a microbial thermotolerance improvement strategy was developed based on adaptive laboratory evolution (ALE) for efficient high-temperature fermentation. Employing this strategy, we have successfully obtained Corynebacterium glutamicum strains with superior resistance to high temperatures. Specifically, the genome analysis indicated that the evolved strains harbored 13 missense genetic mutations and 3 same-sense genetic mutations compared to the non-evolved parent strain. Besides, reverse transcription quantitative PCR analysis (RT qPCR) of the hrcA-L119P mutant demonstrated that both groEL genes were upregulated under 42 °C, which enabled the construction of robust strains with improved heat tolerance. Furthermore, a significant increase in FAS-IA and FAS-IB expression of the fasR-L102F strain was proved to play a key role in protecting cells against heat stress.

Conclusions

This work systematically reveals the thermotolerance mechanisms of Corynebacterium glutamicum and opens a new avenue for revolutionizing the design of cell factories to boost fermentation efficiency.

Background

Microorganisms often suffer serious damage during industrial fermentation due to oxidative and osmotic stimuli, heat, salinity, pH and other environment stresses. Among these stresses, temperature fluctuation during fermentation is one of the most commonly encountered challenges, and it shows strong influences on the solubility of fermentation medium components, the catalytic efficiency of enzymes in metabolic pathways, and the reaction rates of biochemical processes. However, extreme temperatures also have detrimental effects on cell integrity by disrupting the membrane structures or interfering with the functions of biological macromolecules such as proteins and RNAs, thereby reducing cell viability and lowering fermentative capability [1]. Therefore, in order to achieve efficient high-temperature fermentation, the screening and development of robust strains with the desired thermotolerant traits are important prerequisites. Over the past decade, many strategies have been explored to endow industrial strains with improved heat stress tolerance, including adaptive evolution, genome shuffling and rational genetic engineering. In one exciting example of high-temperature fermentation with heat-resistant yeasts, the thermotolerant strain obtained by adaptive evolution or genetic engineering of stress-related genes exhibits a considerable potential for fuel ethanol production from different biomass sources and is superior to the traditional cell factories by saving cooling energy, fermentation time and device space [2, 3].

Microorganisms respond to high temperature by expressing a variety of genes encoding molecular chaperones and heat shock proteins (HSPs), which serve as a universal defense against heat stress [4]. Heat stress can lead to protein misfolding and aggregation after long-term exposure, which is confirmed by a number of studies. By inducing the synthesis of molecular chaperones or heat shock proteins, stress-damaged proteins can be repaired or degraded, thus preventing further damage to microbial cells [5, 6]. Interestingly, a recent report indicated that the introduction of a cold-shock protein (CspL) derived from a thermophilic Bacillus strain also conferred strong high-temperature resistance to living microorganisms such as Escherichia coli and Saccharomyces cerevisiae [7]. Besides, many investigations have revealed that changes in the physical and structural properties of cell membranes also play crucial roles in response to heat shock stress. Adjusting membrane lipid compositions and membrane fluidity, allows microbial cells to improve their heat-resistant properties. However, despite some progress that has been made towards the physiological mechanisms underlying heat resistance in several microorganisms, particularly in E. coli, the development of highly heat-resistant engineered strains still requires further exploration and identification of new potent stress-related genes associated with heat tolerance.

In the biotechnological production of amino acids and organic acids, Corynebacterium glutamicum is known as an indispensable industrial workhorse. Using thermotolerant C. glutamicum strains, high-temperature fermentation can also be utilized to efficiently produce bio-based products like L-glutamate, L-lysine, and L-lactate. From this perspective, the genetically modified strain with excellent physiological robustness against thermal stress is also a crucial guarantee for the improvement of the yield and quality of fermented products.

Consequently, some efforts have been made to investigate the possible mechanisms for heat tolerance in C. glutamicum. For example, genome-wide transcription profiling of C. glutamicum revealed the presence of SigH, HspR, and HrcA regulators that regulated the expression of potential HSP genes in response to heat shock [8]. In another example, in an adaptive evolution of C. glutamicum GLY3 under thermal stress, Oide et al. found that the combination of transgene deletion (pfk and pyk) and missense-mutation expression (glmU and otsA) improved microbial cell growth under thermal stress [9]. Moreover, Kataoka et al. found that the suppression of intracellular potassium leakage at high fermentation temperatures contributed to improving microbial thermotolerance in C. glutamicum [10]. Despite some new findings, the current understanding of heat tolerance mechanisms and the strategies for reinforcing microbial thermotolerance in C. glutamicum are still relatively limited.

In addition, many successful examples of adaptive evolution have endowed C. glutamicum ATCC 13032 with better stress resistance and production performance [11,12,13]. In this study, we sought to develop heat-resistant C. glutamicum ATCC 13032 evolved strains and investigate new genetic variations that assist heat tolerance. The missense point mutations of several genes, especially fasR and hrcA, were found to enable the construction of robust strains with improved heat stress tolerance. HrcA encodes a transcriptional repressor acting on cgR_1436, groEL2, and groES-groEL1, reducing their expression by interacting with the chaperone expression (CIRCE) operator. These genes encode heat shock protein (HSP) molecular chaperones supporting the folding of nascent proteins as well as the repair of damaged proteins in C. glutamicum [8]. The expression of lipid synthesis is controlled by the fasR gene, which encodes a transcriptional regulator of the TetR-type. These findings can facilitate the understanding of the mechanisms that contribute to heat tolerance in C. glutamicum and provide new potential strain improvement strategies related to heat stress tolerance.

Materials and methods

Strains, culture media, and growth conditions

The strains and plasmids employed in this work are listed in Table 1. E. coli DH5α was selected as the competent cells for general cloning. C. glutamicum ATCC 13032 was selected as the parental strain for gene replacement and the wild-type control strain was employed for functional analysis. E. coli cells were routinely grown at 37 °C in LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl), while the C. glutamicum cells were cultivated at 32 °C in LBHIS medium (2.5 g/L yeast extract, 5 g/L tryptone, 5 g/L NaCl, 18.5 g/L brain heart infusion, 91 g/L sorbitol). The media was supplemented with antibiotics at 15 µg/L chloramphenicol or 25 µg/L kanamycin for C. glutamicum. 10 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce gene expression.

Table 1 Strains and plasmids used in this study

Adaptive laboratory evolution

The adaptive laboratory evolution of robust thermotolerant strains was performed by successive shake flask cultivations. In adaptive laboratory evolution under heat stress, C. glutamicum ATCC 13032 was selected as the original parent strain. The evolutionary process was divided into three cycles, including the first 15 passages at 40 °C, the following 15 passages at 41 °C, and the last 45 passages at 42 °C (Fig. 1A). A fresh LBHIS medium was serially inoculated with the evolving cells every 24 h at an initial optical density of 0.1 at 600 nm (OD₆₀₀). In order to select the beneficial thermal-adapted strains, the samples are diluted and spread onto the LBHIS agar plate after each round of passages. The fastest-growing colonies were picked up from the plates and used as the starting strains for subsequent higher temperature evolution. Two independent samples HTMevol-1 and HTMevol-2 were evolved simultaneously by the laboratory experiments of evolution in this study. After adaptation, two individual colonies with the best-performing thermal tolerance were finally isolated from the heat stress screening plates.

Whole genome sequencing and analysis of genetic mutations

Using whole genome re-sequencing, the parental strain C. glutamatium ATCC 13032 and two thermally evolved strains WHT4 and WHT59 were examined for genetic mutations. The genomic DNA of C. glutamicum cells was obtained employing the TIANamp bacterial DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. Whole genome re-sequencing and comparative genomic analysis were conducted in Tianjin Novogene Bioinformatics Technology Co., Ltd. Sanger sequencing was used to identify and validate differences in single nucleotide polymorphisms (SNPs) and insertion and deletion variants (indels) between evolved and parental genomes.

Plasmids and strains construction

The E. coli/C. glutamicum shuttle vector pXMJ-19 was selected as the backbone for the construction of gene expression systems. Using specific primers targeting specific genes, the gene-encoding fragments of the evolved strains containing missense point mutations were amplified. The primers used in this work are shown in Supplemental Table S1. BamHI and XbaI were used to digest the PCR products, which were then ligated into the same sites on pXMJ19 to produce the recombinant plasmids for inducible expression. To study the functional impact of the inducible expression vectors, they were respectively transformed into the wild-type strain C. glutamicum ATCC 13032.

Following the previously described temperature-sensitive plasmid pCRD206, specific genes were replaced by mutations by homologous recombination in two steps. Amplification of genomic DNA of evolved strains was used to introduce genetic variants by amplifying flanking fragments of point mutations in selected genes. The primers employed in these constructions are also shown in Supplemental Table S1. To obtain the corresponding recombinant plasmids, the PCR products were digested with BamHI and XbaI and ligated into pCRD206. By electroporating the plasmid, the wild-type strain C. glutamicum ATCC 13032 was transformed, and two-step selection was utilized to generate allelic replacements. Furthermore, similar strategies were used to create markerless in-frame gene deletion mutants. Sanger sequencing and colony-PCR confirmed the correctness of all mutants.

Quantitative real-time reverse transcription PCR (qRT-PCR)

An RNA prep pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) was used to extract the total RNA of the corresponding strains of C. glutamicum. Following that, Revert Aid first-strand cDNA synthesis kit (Thermo Scientific, USA) was used to synthesize the cDNA. Using a real-time PCR system (Applied Biosystems, USA), 2× SYBR Green Real-time PCR Master Mix (TOYOBO, Osaka, Japan), 0.4 M primers, and the appropriate template DNA were prepared in triplicate, and evaluated with the Applied Biosystems 7500 fast real-time PCR system. The relative fold changes of gene expression were calculated using the delta-delta cycle threshold (ΔΔCt) method based on normalization of 16 S rRNA reference genes.

Cell growth and heat tolerance assays

Before growth experiments were performed, all C. glutamicum cultures were incubated at 32 °C with shaking at 200 rpm for 16 h. We then harvested the culture samples, washed them, and resuspended them in LBHIS medium with a starting OD₆₀₀ of 0.1. To obtain growth curves under normal and heat stress conditions, the OD₆₀₀ values of the tested samples were recorded at different time points. In order to test heat tolerance, the cell suspension was transferred to the fresh preheated medium at a starting OD₆₀₀ of 0.1 and further incubated for 20 h under the indicated heat stress conditions. A minimum of three independent replicates were used to determine growth ability based on final OD₆₀₀ measurements, and error bars are shown for each average.

Fatty acid content assay

Fatty acid determination was carried out using ultra-performance liquid chromatography-mass spectrometry by Beijing Bio-Tech Pack Technology Company Ltd. (Beijing, China) according to the previous reports [14]. In brief, 50 mg of C. glutamicum cells were harvested, washed and resuspended in a 1:1 mixture of isopropyl alcohol and acetonitrile. The extraction process involved vortexing the samples, followed by centrifugation at 12,000 g for 10 min. The concentrations of fatty acids in supernatants were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS), with fatty acids (19:0) as the internal standard.

Results and discussion

Screening of thermotolerant C. glutamicum strains by adaptive evolution

A variety of evidence suggests that the wild-type C. glutamicum cells can grow well over a broad temperature range from 25 °C to 37 °C, while at higher temperatures especially above 40^oC, the overall biomass and metabolic activities of bacterial cells are dramatically reduced. However, these inherent properties limit the application of wild-type C. glutamicum cells in high-temperature fermentations. Recent publications have demonstrated that directed experimental evolution can improve starters by producing more robust strains of C. glutamicum [9]. For the purpose of investigating the mechanisms underlying heat tolerance in C. glutamicum, three successive laboratory evolution cycles with 15 passages at 40 °C, 15 passages at 41 °C, and 45 passages at 42 °C were conducted in order to produce mutant strains that are more thermotolerant. As shown in Fig. 1A, two different heat-adapted strains HTMevol-1 and HTMevol-2 were concurrently evolved from the wild-type ancestor at high temperatures. After being exposed to elevated temperatures, the evolved samples showed enhanced growth compared to the wild-type strain. After finishing evolution, about 100 colonies derived from HTMevol-1 and HTMevol-2 were picked into 96-deep-well plates for further re-screening, and two fastest-growing individual colonies named WHT4 and WHT59 were obtained. Growth assays at different temperatures revealed that the two adapted strains showed markedly improved tolerance toward higher temperatures above 40 °C compared with the non-adapted strain (Fig. 1B). When cells were grown at 42 °C, the biomass of two adapted strains was approximately 2.5-fold higher than that of wild-type strains. These heat tolerance data were further confirmed by cell growth curves at 32 °C and 42 °C, in which the adapted strains WHT4 and WHT59 were more tolerant to higher temperature than the ancestral wild-type strain (Fig. 1C).

Fig. 1

ALE of C. glutamicum under thermal stress. (A) Culture conditions and scheme of the entire evolution experiment. Daily serial transfer of cell culture was performed at the exponential growth phase. Cycles 1–3 represent the two strains at 40 °C, 41 °C, and 42 °C. (B) Thermal growth characteristics of the bacterial strains acquired from the evolution experiment. (C) Cell growth curves of the bacterial strains acquired from the evolution experiment

Comparative genomic analysis for investigating stress-related genetic variations

To explore candidate genes and variants that confer high heat resistance, we, therefore, performed whole-genome resequencing of two thermal-evolved mutants and their parental wild-type strain. As compared with the laboratory-stocked wild-type strain, these evolved strains had 23 point mutations and 3 genomic insertions/deletions, which was demonstrated through the comparative genomic analysis. The re-examination of potential mutations by Sanger sequencing, however, failed to detect all three insertions/deletions, suggesting that these data were intended as sequencing errors. Among all the observed point mutations, a total of 16 point mutations were observed in the coding sequence (CDS) region, and 7 mutations were found outside of the CDS. As shown in Table 2, of the 16 intragenic point mutations in the evolved strains, 13 amino acid substitutions were nonsynonymous, and the rest 3 genetic mutations were synonymous.

Table 2 Amino acid substitutions observed in the evolved strains

The evolved strains showed point mutations in 13 missense sequence variants including rp1D, cg0892, cspB, gltA, leuC, cg2066, cg2106, hrcA, cg2614, fasR, cg3078, and dnaK, suggesting they may be involved in thermal adaptation. Based on the genome annotation of C. glutamicum in the KEGG and NCBI database, rplD encodes a 50 S ribosomal protein L4 that is response for ribosome assembly; cspB encodes a cold shock-like protein that can regulate the expression of cold shock genes in response to cold stress; gltA encodes citrate synthase involved in the tricarboxylic acid (TCA) cycle; leuC encodes 3-Isopropylmalate dehydratase that is essential to the leucine biosynthesis; hrcA encodes a heat-inducible transcription repressor that plays a key role in negatively regulating the expression of heat shock genes like grpE-dnaK-dnaJ and groELS operons; fasR encodes a TetR-type transcriptional regulator that controls the expression of lipid synthesis; dnaK encodes heat shock protein hsp70 that can be regarded as a chaperone. Unfortunately, due to the lack of genome annotation information, the potential functions of remaining 5 genes including cg0892, cg2066, cg2106, cg2614, and cg3078 were still unclear.

Determination of beneficial point mutations conferring heat resistance

Fig. 2

Growth assays of the wild-type and specific point mutant strains based on the pXMJ-19 plasmid (A) or genomic mutagenesis (B), (C) in response to thermal challenges. Data represent mean values from three independent cultures, and the standard deviation from the mean is indicated as error bars

To identify the possible beneficial point mutations that facilitate cell growth under thermal stress, we constructed a serial of recombinant plasmids containing different missense point mutations and transformed them into the parental wild-type strains for growth assays. A comparison of the growth abilities of all tested strains harboring different types of point mutations at 32 °C was shown in supplemental Figure S2. The strains that contained specific mutations in cspB, gltA, cg2066, hrcA, fasR, and cg3078 displayed a clear increase in microbial growth at 42 °C, as illustrated in Fig. 2A. The above six positive mutations were introduced into the genomic DNA of the parental wild-type strains in order to further investigate the role of these residues in heat resistance. In a study by Nishio and colleagues, 40 °C was reported as the maximum temperature at which C. glutamicum can grow [15]. Growth assays supported that the direct introduction of fasR-L102F or hrcA-L119P mutation into wild-type genomic background obviously contributed to increased heat resistance at elevated temperature (Fig. 2B). In particular, the beneficial strain carrying the fasR-L102F mutation showed a 38% increase in cell growth as compared with the wild-type counterpart at 42 °C. Furthermore, when the two beneficial point mutations in the genome were introduced concurrently, microbial biomass was significantly increased by 55.6% over wild-type control during heat stress (Fig. 2C). This robust augmentation provides further corroboration for the pivotal roles of the hrcA-L119P and fasR-L102F mutations in conferring thermal resistance.

Effects of hrcA-L119P mutation in the regulation of heat-shock response

Fig. 3

Response mechanism to heat shock exhibited by the hrcA-L119P. (A) Schematic drawing to illustrate hrcA regulatory mechanism in C. glutamicum. (B) Relative mRNA levels of the heat shock protein (HSP) genes encoding molecular chaperones in wild-type ATCC 13032 and hrcA-L119P mutant at 32 °C and 42 °C. Total RNAs were prepared from cells grown to the early exponential phase in LBHIS medium. Aliquots of RNAs were reverse transcribed and subjected to qPCR. The transcript levels in wild-type ATCC 13032 were set to 1.0. Data represent mean values from three independent cultures

The heat shock response efficiently safeguards cells and organisms from adverse environmental conditions including physiologically elevated temperatures, oxidative stress, cytotoxic agents, etc. All organisms universally increase expression of highly conserved proteins known as heat shock proteins (HSPs) in response to sudden increases in temperature. The molecular chaperones help fold newly synthesized proteins and repair damaged proteins by aiding in folding them. Genes of groES-groEL1 and groEL2 encode heat shock proteins (HSPs) that act as molecular chaperones in C. glutamicum [8]. HrcA, as far as we know, is a transcriptional repressor that negatively regulates stress response genes through its interaction with conserved inverted repeat elements called CIRCEs (Fig. 3A). Sequence analyses of groES, groEL2, and dnaK promoters enable the identification of CIRCE elements close to the promoter’s -10 and − 35 regions in C. glutamicum [8]. A potential role for HrcA in the regulation of the heat shock response in C. glutamicum has been suggested by this observation.

Based on the growth experiments described above, hrcA-L119P mutant has distinct consequences for growth at 42 °C. The expression profiles of several heat shock protein families were examined to further elucidate the molecular basis of hrcA-L119P mutation and the regulation of heat shock response (Fig. 3B). It is shown that, regardless of the temperature (either 32–42 °C), the introduction of hrcA-L119P mutation led to an obvious increase in transcript levels of several HSP genes such as groEL2, groEL and dnaK. Notably, hrcA-L119P mutant strains expressed approximately 3.6-fold more groEL2 than wild-type strains. Despite the marginal reduction in transcript levels for the other two HSP genes (groES and groEL1) under normal conditions, a discernibly elevated transcript level was observed in response to increased temperature. Furthermore, compared with the wild-type controls, the HcrA-L119P mutant strain showed a more significantly up-regulated heat shock protein gene expression levels under high temperature conditions. Ehira’s study demonstrated that the disruption of the hrcA gene resulted in increased transcript levels of groES, groEL1 and groEL2 at 33 °C, with elevations of 2.7-fold, 2.90-fold and 3.13-fold, respectively [8]. Similar results were obtained by Barreiro, who found that the transcript levels of groES, groEL1 and groEL2 were elevated by 3 to 3.5-fold at 30 °C by the disruption of hrcA gene [16]. Compared with the hrcA disruption strain, we found that the hrcA-L119P mutant strain mainly upregulated the expression level of heat shock protein genes including groEL2, groEL and groEL1 under high temperature conditions, which may help to avoid the persistent and irreversible effects of a direct knockout on the strain’s physiological state.

It is the co-repressor (GroEL) rather than drastic changes in HrcA itself that influence the regulation of the HrcA/CIRCE system [8, 16]. According to recent study, Escherichia coli can grow at 48.5 °C only with exceptional high GroEL and GroES levels, despite a permanently induced heat shock response [17]. In our study, the hrcA-L119P mutant showed good growth ability at 42 °C, mainly due to that both groEL genes showed upregulation in hrcA-L119P mutant at 42 °C. The expression of groEL2 increased the most at both 32 °C and 42 °C. The expression of groES is similar to the expression of groEL1 since groES and groEL1 form a bicistronic operon in the genome of C. glutamicum, whereas groEL2 is expressed as a monocistronic transcript [16].

In C. glutamicum, the chaperone DnaK encoded by dnaK promotes proper protein folding, preventing poorly folded proteins from aggregating. As the HrcA regulator binds to the CIRCE motif in the groES-EL and dnaK operons of B. subtilis, the operons are negatively controlled by the HrcA regulator [18]. Nonetheless, the dnaK operon of C. glutamicum encodes its autoregulatory repressor designated HspR/HAIR (HspR-associated inverted repeat) system and is positively controlled by the alternative sigma factor the σH/σE [16, 19]. In our study, the transcript levels of dnaK was elevated at both 32 °C and 42 °C in the hrcA-L119P mutant. The reasons behind this phenomenon require further investigation.

Meanwhile, Oide’s study showed that mutations in hrcA did not affect heat tolerance, whereas our study found that mutations in hrcA had a significant effect on heat tolerance in C. glutamicum because the missense mutation which was caused by the substitution of a leucine residue with a lysine residue at 119 (L119P) is the causative mutation for heat tolerance.

fasR-L102F mutation improves heat tolerance by regulating fatty acid synthesis

Fig. 4

Response mechanisms to heat shock exhibited by the fasR-L102F. (A) Schematic drawing to illustrate fasR regulatory mechanism in C. glutamicum. (B) Relative mRNA levels of the selected fatty acid biosynthesis genes in wild-type ATCC 13032 fasR-L102F and ΔfasR mutant at 32 °C and 42 °C. Total RNAs were prepared from cells grown to the early exponential phase in LBHIS medium. Aliquots of RNAs were reverse transcribed and subjected to qPCR. The transcript levels in wild-type ATCC 13032 were set to 1.0. Data represent mean values from three independent cultures. (C) Fatty acid content by fasR-L102F and ΔfasR. Data represent mean values from three independent cultures, and the standard deviation from the mean is indicated as error bars

According to the growth experiments described above, the fasR-L102F mutant causes distinct growth consequences when grown at 42 °C. The molecular basis of this phenotype should be further elucidated. Considering the fasR-L102F mutation identified in C. glutamicum was related to fatty acid biosynthesis genes transcriptionally regulated by the TetR-type transcriptional regulator FasR (encoded by fasR gene), it was hypothesized to be a fatty acid biosynthesis-related mutation. A qRT-PCR was performed to quantify gene expression in the fasR-L102F mutant in order to determine whether lipid synthesis-related genes were differentially expressed (Fig. 4A). Furthermore, C. glutamicum was also modified to eliminate the gene fasR to investigate how the FasR protein regulates gene expression.

As shown in Fig. 4B, in both 32 °C and 42 °C experiments, cg0812 (accD1, dtsR1) transcript levels increased over eight-fold in ΔfasR, whereas the accBC, fasIA, and fasIB transcript levels were influenced by temperature. Transcription of the accBC, fas-IA, and fas-IB genes increased significantly in ΔfasR only when heat stress was applied. The fasR-L102F mutation resulted in increases in the transcript levels of fasIA and fasIB at 42 °C by 2.13-fold and 4.09-fold, respectively, whereas the mutation did not affect the expression of accBC and accD1. In contrast, fasIA and fasIB genes’ transcription was lower than the control in fasR-L102F and ΔfasR at 32 °C. In addition, the accBC gene showed a stronger upregulation in ΔfasR at 42 °C, but not in fasR-L102F. In a word, the results obtained suggest that FasR regulates lipid metabolism genes in an important way.

In C. glutamicum, acetyl-CoA carboxylase, composed of the three polypeptides AccD1, AccBC, and AccE, synthesizes malonyl CoA, the substrate of the FA synthases [20, 21]. C. glutamicum has two FAS-I enzymes. The essential FAS-IA enzyme is responsible for stearate and oleate synthesis, while FAS-IB is predominantly responsible for palmitate synthesis [22]. FasR is a widely distributed regulator that controls cellular fatty acid synthesis, repressing the genes for acetyl-CoA carboxylase, Fas-IA and Fas-IB with the effector acyl-CoA [23]. In the mRNA quantifications at 42 °C, accD1, accBC, fasIA and fasIB transcript levels were greatly elevated due to the deletion of fasR, while other genes were little affected, in agreement with Nickel’s research [23]. In this study, the expression of cg0812 is a compensation for FasR deletion and is not affected by temperature. However, fasIA transcript was influenced by temperature. Transcriptional regulator FasR binds to five upstream regions of the transcription factors accD1 and fasIA [24]. In a typical manner of TetRs, FasR binds directly to the promoter region of accD1 and fasA to prevent transcription [23]. Other regulators likely play a role in transcription regulation. The result showed that mutation from leucine to phenylalanine at position 102 of fasR resulted in significant disruption in fasR gene function. The missense genetic mutation of fasR caused a significant increase in FAS-IA and FAS-IB expression, which was the main reason for the growth enhancement of the fasR-L102F strain at high temperature.

C. glutamicum’s strong differential expression of cg0812, fasIA, and fasIB in both ΔfasR and fasR-L102F led us to verify the total lipid content. It was found that the fatty acid content of ΔfasR at 32 and 42 °C was significantly greater than that of the control. As previously reported, the loss of fasR function is required for C. glutamicum to overproduce fatty acids [25]. As a result, the fatty acid content of fasR-L102F at 32 °C was 6.32 times greater than that of the control, and it was 14.23 times greater at 42 °C. It was higher than that of the control yet lower than that of ΔfasR (Fig. 4C). Changes in the relative content of fatty acids in cell membranes at high temperature play an important role in cell growth [26]. The increase in fatty acids can increase cellular fluidity, which might benefit cells by providing resistance against thermal stress.

Conclusions

This work demonstrates that experimental evolution conducted in an increasingly thermal environment has paved the way for exploring the gene functions of C. glutamicum and identified promising candidate genes to further understand the bacterium’s thermotolerance mechanisms and enhance its heat resistance. Significantly, we have revealed that the hrcA-L119P and fasR-L102F mutations play a pivotal role in enhancing microbial thermotolerance, providing crucial guidance for the development of a universal temperature stress resistance strategy. The construction of novel temperature-tolerant C. glutamicum is crucial for enhancing the fermentation efficiency and yield in high-temperature fermentation processes, holding significant importance for industrial-scale production of amino acids, organic acids and riboflavin.