Metabolic and evolutionary engineering, and the growth rates of mutant strains
The ldh and poxB genes, which are main contributors of the by-products acetate and lactate [21], were deleted. Additionally, the methylmalonyl-CoA carboxyltransferase (mmc) gene from P. acidipropionici¸ which controls the carbon flow into the propionate-producing Wood-Werkman cycle [22] was overexpressed. These manipulations yielded the strains P. acidipropionici-Δldh, P. acidipropionici-Δldh-ΔpoxB, and P. acidipropionici-Δldh-ΔpoxB + mmc, respectively. In evolutionary engineering, specific stress is applied gradually on the basis of the growth rate of the adapted strain, which gives a buffer period so that the strain can adequately mobilize the in vivo response mechanisms to resist stress (Fig. 1). During the evolution of P. acidipropionici-Δldh into the mutant I, the growth of the experimental strain displayed a peak at OD600 17.1 on the fourth day of culture, followed by a downward trend because of the decline of pH and the increase of glucose concentration. Subsequently, the growth rate of P. acidipropionici-Δldh tended to be stable at OD600 13.9 and began to descend when the pH was decreasing again (6 to 5), and was ultimately stable at OD600 15.9. From P. acidipropionici-Δldh-ΔpoxB to the mutant II (deletion of poxB followed by evolutionary engineering), the formation of the subsequent two peaks at OD600 16.5 and 15.4 on the 6th and 15th day corresponded to the progressive deepening of the levels of single stress factors. From P. acidipropionici-Δldh-ΔpoxB + mmc to the mutant III (overexpression of mmc followed by evolutionary engineering), the growth rate of the strains started to pick up a steady upward trend, and the strain showed a normal growth curve after supplementation with fresh medium. The highest OD600 was 14.6, while the lowest OD600 of 9.8 was observed with pH 4, 120 g·L− 1 glucose and 21% oxygen sparging. These results indicate that the experimental strain could still maintain a considerable growth rate under the superimposed cross stress, even when exposed to simultaneously increasing stress levels. This indicated that the strain had developed a certain level of ‘cross-stress’ protection and acquired a fitness advantage based on the gradual application of stress by artificial control so that it could deal with cross stress.
To further investigate the growth rate of the strain obtained by metabolic and evolutionary engineering, the wild type and mutant III were compared. Compared with the 0.09 h− 1 specific growth rate of the wild type under normal fermentation conditions (pH 7), mutant III had a significant improvement at 0.14 h− 1, which was apparently higher than the 0.131 ± 0.007 h− 1 previously reported for growth on soy molasses and soy molasses hydrolysate [2]. However, when the strains were exposed to low pH, the growth of both the wild type and the mutant III was suppressed accordingly. Nevertheless, the growth of the mutant III was significantly superior to that of the wild type. With the decrease of pH, the growth rate difference between the wild type and mutant III gradually increased, and mutant III could maintain normal growth, while the growth of the wild type was clearly suppressed (Fig. S1A). As the concentration of glucose in the medium increased, the difference of the growth rate between the wild type and mutant III gradually increased. At normal glucose concentrations (30 g·L− 1 glucose), the specific growth rate of the wild type was about 0.09 h− 1, while that of mutant III reached 0.2 h− 1, which was 1.4-fold higher than the previously reported value for the same glucose concentrations [23]. While the wild type almost stopped growing at a high glucose concentration of 120 g·L− 1, mutant III could maintain growth at OD600 0.4 (Fig. S1B). Futhermore, the strain exhibited similar growth characteristics when exposed to oxygen stress compared with acid stress and osmotic stress. A 2-fold growth rate difference has observed under anaerobic fermentation conditions (oxygen content 0.001%). Under these conditions, the specific growth rate of mutant III reached 0.18 h− 1, while that of the wild type was 0.09 h− 1. By contrast, with 21% oxygen content in the sparged gas mixture, the specific growth rate of the wild type was only 0.03 h− 1, while that of mutant III was 0.05 h− 1 (Fig. S1C). As a result, the growth performance of mutant III under cross stress was clearly superior to that of the wild type. Importantly, the generation of the mutant III has proved that evolutionary engineering is an effective method for improving the resistance of P. acidipropionici to cross stress.
PA fermentation using the engineered P. acidipropionici
Batch fermentations were carried out using wild-type P. acidipropionici as well as the mutants I, II and III to investigate their PA production capacity. As shown in Table 1, under the optimal culture conditions (pH 7, 99% N2, 5 g·L− 1 glucose), the PA titer and productivity of mutant I were increased by 12.4 and 13.5% compared to the wild type. Its performance was therefore also significantly better than that of P. jensenii-Δldh with 1.6 and 8.1% enhancement [11], respectively. At the same time, a 68.8% of decrease of LA yield (3.06 ± 0.82 vs. 1.04 ± 0.17 g·L− 1) was obtained. However, there was a 3% increase of AA titer (5.24 ± 0.83 vs. 5.63 ± 0.62 g·L− 1), which may be related to the deletion of ldh, which increased carbon flow from pyruvate into the synthesis of LA [21]. The PA production of the mutant II only increased by 3.2%, while the titers of AA and LA were both decreased by more than 70%, which was consistent with the variation tendency of the PA, LA and AA titers of P. acidipropionici-ΔpoxB-Δldh [17]. A further 37.1% increase of PA titer (28.1 ± 0.96 g·L− 1 vs. 38.7 ± 1.14 g·L− 1) and 37.8% increase of PA productivity (0.216 ± 0.006 g·L− 1 vs. 0.298 ± 0.008 g·L− 1·h− 1) was achieved in the mutant III. This increase was much more significant than the reported value achieved via the overexpression of mmc in P. freudenreichii in batch fermentation [23]. At the same time, there was a 78.6 and 87.8% decrease of the byproduct yields because of the deletion of ldh and poxB, and the overexpression of mmc drove the carbon flow into the synthesis of PA to the greatest extent.
Table 1
Analysis of microbial production using engineered P. acidipropionici in batch fermentation.
Strain | PA titer (g·L − 1) | LA titer (g·L − 1) | AA titer (g·L − 1) | PA productivity (g·L − 1·h − 1) |
WT | 28.1 ± 0.96 | 3.06 ± 0.82 | 5.24 ± 0.83 | 0.216 ± 0.006 |
Mutant Ⅰ | 31.8 ± 0.85 | 1.04 ± 0.17 | 5.63 ± 0.62 | 0.245 ± 0.007 |
Mutant Ⅱ | 29.4 ± 0.59 | 0.89 ± 0.06 | 0.78 ± 0.04 | 0.226 ± 0.006 |
Mutant Ⅲ | 38.7 ± 1.14 | 0.78 ± 0.05 | 0.69 ± 0.05 | 0.298 ± 0.008 |
Comparative omics analysis of the wild type and the mutants I, II, III
The adaptation of microbes to environmental stress can produce beneficial phenotypes through random genomic mutations and subsequent positive selection [24]. In this study, the resistance mechanism of the strains to cross stress may be elucidated by analyzing the transcriptome, proteome and metabolome of the different P. acidipropionici strains. The original reads of transcriptome and proteome have been deposited in the NCBI Sequence Read Archive (Accession No. SRR10597965) and PRIDE database (Accession No. PXD016616), respectively. The results showed that the three evolved mutants reflected the dynamic evolution of the resistance mechanism of P. acidipropionici based on the wild type. At the transcriptional level, the differentially expressed genes (DEGs) predicted in the three groups compared to the wild type are shown in Fig. 2a. A total of 166 DEGs were identified in the mutant I, with 72 up- and 94 down-regulated genes. Similarly, 165 DEGs were identified in mutant II (85 up- and 80 downregulated genes) and 102 in mutant III (29 up- and 73 downregulated genes). The MA plot and volcano plot (Fig. 2b) show the distribution of the DEGs between the two groups of samples. As can be seen in the Venn diagram (Fig. 2c), there were 50 identical genes that were differentially expressed, meaning that a considerable number of shared or similar metabolic pathways were shared between the three mutants and the wild type. A total of 2,158 proteins were identified in the proteomic analysis (Fig. 2d). There were 343, 260, and 226 differentially expressed proteins (DEPs) in the mutants I, II, and III, respectively. Among these, 217, 176, and 147 proteins were up- while 126, 84, and 79 were downregulated, respectively. Likewise, we discovered that 72 proteins involved in the same function were differentially expressed in the three mutants. The Principal component analysis (PCA) score plot was performed to show the intracellular metabolism differences of the wild type and the mutant III (three parallel samples in each group) [25]. As shown in Fig. 3a, the strains obtained were clearly divided into four groups and the data sample point dispersion of four groups was relatively great, which indicating the metabolic profile of four groups was significantly different. Figure 3b revealed 34 significantly differentially abundant intracellular metabolites, which were mainly involved in the three central metabolic pathways, including glycolysis (D-glucose, trehalose), the tricarboxylic acid cycle (malic acid), the pentose phosphate pathway (phosphoric acid), and amino acid metabolism (glycine, threonine and serine) [26]. There were a total of 17 up- and 10 downregulated metabolites in mutant I, 21 up- and 13 downregulated metabolites in the mutant II, as well as 21 up- and 12 downregulated metabolites in mutant III. Combined with the sample correlation analysis (Fig. 4), the data revealed that mutant I had the lowest similarity with the wild type, while mutant III had the highest. From the perspective of difference, mutant I showed the most dramatic changes and the most intense responses to cross stress, while mutant III was the opposite.
Based on the transcriptome sequencing results, the GO enrichment analysis of the DEGs was conducted as shown in Fig. 5. DEGs involved in the categories metabolic process, single-organism process in biological process, membrane and membrane part in cellular component, catalytic activity, binding and transporter activity in molecular function were all enriched with false discovery rate (FDR) q-values of less than 0.05 in the three mutants compared with the wild type. Among them, the membrane part and catalytic activity were significantly different. This suggested that the DEGs involved in these items were positively responsive, especially the major annotated categories membrane part and catalytic activity, which were most significantly affected by the cross stress. Moreover, it is worth noting that the DEGs were only downregulated related to transporter activity in the mutant III. The adverse cross stress conditions impacted 97 KEGG pathways in the mutant II, 84 KEGG pathways in the mutant I, and only 44 KEGG pathways in the mutant III. The top 20 enriched KEGG pathways (corrected P-value < 0.05) were displayed in the scatter plot (Fig. 6). Compared with the wild type, genes related to carbon fixation pathways involving energy metabolism were upregulated in the mutants I and II, while they disappeared from the data of mutant III. Interestingly, only five metabolic pathways with upregulated genes were enriched in the mutant III, indicating that this mutant has become a mature, highly tolerant mutant on the basis of the mutants I and II. Therefore, in order to adapt to the high glucose, low pH and the aerobic fermentation environment, the mutants increased their tolerance ability to environmental stress by changing the gene expression of the relevant metabolic pathways to maintain growth and metabolism under environmental stress.
The distribution of DEPs quantified in the GO secondary annotation was statistically analyzed to gain insights into the potential functional implications for stress tolerance (Fig. 7). The categories metabolic process (46.88%, 51.72%, 38.81%) in biological process, membrane (31.17%, 27.27%, 35.48%) in cellular component, as well as catalytic activity (56.99%, 61.90%, 58.52%), and binding (33.16%, 29.93%, 27.41%) in molecular function were enriched in the mutants I, II and III, respectively, which was roughly consistent with the GO enrichment analysis of the DEGs. However, the mutant I no longer showed the most obvious changes as was the case at the transcriptome level. This result suggested that major functional categories of the strain involved in regulation remained largely unchanged under adverse environmental conditions, both at the gene and protein levels. To further determine the DEPs more likely to be mobilized to resist stress, we enriched the DEPs. In the GO enrichment (Fig. 8A), we observed that upregulated DEPs were involved in glutathione transferase activity and FAD binding in the mutant I, glycerol metabolic process and alditol metabolic process in the mutant II, while they were involved in cysteine biosynthetic process and sulfate reduction in the mutant III. In the KEGG enrichment (Fig. 8B), the microbial metabolism in diverse environment-related pathways were enriched (1.64-fold, 1.79-fold, 1.66-fold) in the mutants I, II and III, respectively. Degradation of aromatic compounds and bacterial chemotaxis were detected to be significantly enriched in the mutants I and II, while sulfur metabolism was found in the mutant III, demonstrating that this mutants developed positive responses to the adverse environment. As can be seen in Fig. 9, at the metabolic level, carboxylic acids metabolism such as butanoate and pyruvate, methane metabolism, amino acid metabolism such as glycine, serine and threonine, as well as cysteine and methionine in the three mutants were all significantly affected when subjected to cross stress. The pathway analysis results implied the mutants might modulate the stress response mechanisms against to abiotic stress.
Differentiation analysis of the determinants related to cross stress of the wild type and the mutants I, II and III
In order to understand the detailed resistance mechanism of P. acidipropionici subjected to cross stress, we investigated the variation footprints of the changes from the wild type to mutant III, from the DNA to metabolite levels. Maintaining pH homeostasis is the main challenge under acid stress, which may be supported by ATP metabolism and active transport of protons. The ΔpH in the cell can drive the generation of proton motive force (PMF) to enhance cellular protonation [27]. Previous studies have shown that specific genes are commonly expressed by Gram-positive neutrophilic bacteria during acid stress [28]. In this study, genes related to the ATPase-coupled transmembrane transporter activity were confirmed to be downregulated to retard protonation based on the transcriptome analysis in the three mutants. The interconversion between NADH and NAD is associated with ATP metabolism in the coupling of proton levels. The NADH levels were reduced 2.97-, 2.80-, and 3.54-fold, while those of NAD were reduced 8.11-, 6.50-, and 5.81-fold in three mutants, respectively. Moreover, the determinants of the regulation of pH homeostasis, such as cations and inorganic ions, were also discovered in the three mutants. ABC transporters were transcriptionally upregulated in the three mutants, which may mediate the translocation of large amounts of substrate across the cell membranes [29]. The Dps protein, which protects cells against acid damage [30], was expressed 3.47-fold more at the transcriptional level in the mutant I, as well as 2.15-, 1.89-, and 3.49-fold more at the proteome level in three mutants compared to the wild type, respectively. The genes GroES and dnaK related to the acid adaptation of bacteria were also differentially expressed at the protein level [31]. GroES were upregulated in all three mutants. DnaK was downregulated in mutant I, but upregulated in the mutants II and III. Furthermore, GroES was found downregulated in the mutants I, II according to transcriptional analysis. Amino acid metabolism plays an important role in the acid tolerance of bacteria [32]. The arginine deiminase (ADI) system, which consists of arginine deiminase, carbamate kinase (CK) and ornithine transcarbamoylase, has been proved to exist in the genus Propionibacterium [33, 34]. This system is activated under acidic conditions because of its contribution to pH homoeostasis [28]. In this study, the expression of ADI and CK were not detected in RNA-seq and metabolome analysis, but the proteins were detected in the proteome. Moreover, the glutamate decarboxylase (GAD) pathway, which can consume H+ [35], did not show significant differential expression in the mutant I, II, and III, respectively. Therefore, the trajectory of change of ADI and GAD remained consistent, whereby mutant II showed more positive regulation of ADI and GAD, while mutant I was more positive in the regulation of CK.
We observed that the ABC-type glycine betaine transport system, which is related to the adaptability to osmotic stress [36], was not observed transcriptionally regulated in the three mutant, but upregulated 2.81-, 2.06-, 4.51-fold on the proteomic level in the mutant I, II, and III. Additionally, it was reported that high osmolarity can induce the oxidative stress regulons [37, 38]. It has been reported that H2O2 can be produced by NADH oxidase in the cell membrane when anaerobes are exposed to oxygen, posing a threat to cell survival [39]. Interestingly, we only observed a significant upregulation of NADH oxidase by proteome analysis with 2.00-, 2.05-, 2.05-fold in three mutants. In addition, the expression of superoxide dismutase (SOD) and peroxidase (POD) was upregulated in the protein level. Notably, there was a 2.99-fold upregulation of SOD expression in the mutant III and 3.18-fold upregulation of POD expression in the mutant II. These results indicate that the mutants also adapted to oxidative stress. In addition, as a bioprotective agent, trehalose exhibits prominent biological functions against adverse abiotic stress, such as oxygen radicals and acid stress [6]. Found by metabolomics, the level of trehalose was a persistent upward trend from the wild type to the mutant III, especially the mutant III with the most significant level in the content compared to the wild type. It verified that the mutant III was subjected to the relatively large degree of cross stress. Some metabolites such as serine, aspartic acid, glycine and trimethylol propane were only observed in the mutant II and III, while methanediimine, glycol acid and ethylaminewhich were only observed in the wild type.
Pyruvate from glucose or glycerol is a vital precursor in the metabolic pathway of propionic acid synthesis in Propionibacterium spp. However, the pyruvate is also converted into byproducts, most importantly into acetate by poxB and lactate by ldh [18]. Therefore, poxB and ldh were selected to be deleted to reduce the metabolic flux into the formation of acetate and lactate. However, another source of acetate synthesis is the conversion of pyruvate to acetyl-CoA. This indicated that the deletion of poxB did not completely block the carbon flux into acetate synthesis, which was also confirmed by the fermentation data. Unsurprisingly, no differences of ldh and poxB were found in the omics results. The lactate permease involved in lactate transport driven by a proton motive force was respectively upregulated 3.6-, 2.6- and 1.88-fold in the mutants according to the proteomic analysis. Therefore, decreased lactate synthesis promoted lactate permease activity to maintain the intracellular metabolic homeostasis [40]. For PA synthesis, pyruvate is converted to oxaloacetate, which finally flows to PA via mmc through the transfer of one carbon from methylmalonyl-CoA to pyruvate [41]. The overexpression of mmc led to 1.12- and 2.73-fold, as well as 1.34- and 2.58-fold upregulation in the mutant II and III according to the RNA-Seq and proteomic analysis, respectively. However, there were no differences in mutant I, indicating that the increase of carbon flux to PA formation due to the deletion of ldh could be neglected. This was corroborated by the fermentation results. Moreover, succinate dehydrogenase converts fumarate to succinate, which is also an important precursor of PA. It was found to be 2.88- and 2.63- upregulated at the transcriptional level in the mutant I and II, as well as 2.94-, 2.29- and 1.85-fold at the protein level in the three mutants, respectively. Interestingly, pyruvate carboxylase, which converts pyruvate to oxaloacetate, likewise was only found to be upregulated in mutant III, 1.66-fold at the protein level, which explains why the omics results for mmc and succinate dehydrogenase were inconsistent. Moreover, the variation trend of the contents of PA, LA and AA at the metabolic level from the wild type to the mutant III was consistent with the fermentation results.