We found that Pseudomonas putida KT2440 can adapt to acidic environments, maintaining viability and metabolic activity at pH levels as low as 4.5. This acid tolerance is crucial for industrial applications where pH fluctuations occur.
The acidity of vinasse was examined under different solid–liquid ratios. Results showed that within the range of 1:5 to 1:30, the pH value remained stable between 4.3 and 4.6, indicating that the environment of vinasse is generally acidic and imposes significant stress on the growth of Pseudomonas putida KT2440.
Cultivation under various pH conditions revealed that Pseudomonas putida KT2440 grew well in neutral and near-neutral environments. When pH was greater than 5.0, the strain could still grow but was markedly inhibited compared with neutral conditions, whereas growth was almost completely suppressed under pH values below 4.5. Based on the acidity of vinasse, the adaptation target was set as improving the growth performance of Pseudomonas putida KT2440 at pH 4.5.
By gradually reducing the culture pH, a strain capable of stable growth at pH 4.5 was successfully obtained. Compared with the non-adapted strain, the acid-tolerant strain exhibited significantly improved growth curves under acidic conditions, with a trend similar to that under neutral conditions, except for a slightly lower cell density at the stationary phase. Moreover, the acid-tolerant strain was able to form colonies on both acidic solid and semi-solid media, further confirming its ability to grow in acidic environments.
The adapted strain caused a significant increase in the pH of the culture medium under different conditions: from approximately 7.2 to 8.7 in neutral LB and from about 4.8 to 8.6 in acidic LB. These findings indicate that the acid-tolerant strain alleviates acidic stress by actively shifting the surrounding environment toward alkalinity, thereby achieving adaptation to acidic conditions.
Table 1. Medium pH before and after inoculation with acid-tolerant Pseudomonas putida KT2440
| Condition | Initial pH | Final pH (Rep 1) | Final pH (Rep 2) |
|---|---|---|---|
| Normal LB | 7.17 | 8.77 | 8.76 |
| Acidic LB | 4.75 | 8.53 | 8.68 |
The genetic engineering operations performed on wild-type Pseudomonas putida KT2440 successfully established and validated the core functional modules required for enhanced succinate production. Through precise knockout of the sdhA gene and heterologous expression of the dcuB transporter, we confirmed the individual and combined functionality of these genetic elements in a standardized, well-characterized laboratory strain. This foundational work served as critical proof-of-concept, de-risking the subsequent engineering of the more complex acid-tolerant variant by demonstrating that the “Block” and “Secrete” strategies are functionally compatible and effective in the KT2440 chassis.
Phenotypic characterization under controlled fermentation conditions provided compelling evidence for the functional efficacy of the engineered modules. Comparative analysis revealed several key findings:
Essential role of export systems: The absence of significant difference in extracellular succinate between WT and ΔsdhA strains demonstrates that KT2440 lacks native succinate efflux capacity, and intracellular accumulation through consumption blockade alone is insufficient to enhance extracellular titers.
Functional validation of heterologous transporter: Introduction of dcuB into the wild-type background (WT+D4/D6) resulted in markedly increased extracellular succinate concentrations compared to WT , confirming successful functional expression and activity of the heterologous transporter.
Synergistic module interaction: The highest succinate secretion was consistently observed in ΔsdhA+D4/D6 strains , demonstrating that combining the consumption blockade (“Block” strategy) with enhanced export (“Secrete” strategy) creates a powerful synergistic effect that maximizes succinate production.
Efficient constitutive expression: The minimal difference between induced (+I) and non-induced (-I) conditions across most strains suggests substantial basal expression from the vector system, providing sufficient transporter activity without requiring precise induction control.
Through multiple rounds of sequential genetic engineering, we successfully integrated the heterologous expression of E. coli-derived fumarate reductase gene (frdA), the succinate efflux pump gene (dcuB), and the knockout of the succinate dehydrogenase gene (sdhA) into an acid-adapted Pseudomonas putida KT2440 chassis. This resulted in the final engineered strain, Acid-Tolerant KT2440 (ΔsdhA / pBBR-AmpR-frdA / pBBR-KanR-dcuB), with its key genetic constructs and validated function in the native host confirmed.
Phenotypic analysis under acidic fermentation conditions (pH ~4.5) demonstrated that the final engineered strain exhibited remarkable succinate production capacity. The strain possessing the combined sdhA knockout, frdA, and dcuB expression consistently achieved significantly higher succinate yields compared to all control groups, in acid-tolerant genetic backgrounds, validating the success of our metabolic engineering strategy.
We constructed a recombinant plasmid containing the T. versicolor laccase fused with EGFP, guided by its native signal peptide of the laccase. After transforming the plasmid into Pichia pastoris and inducing expression with methanol, we used a confocal microscope to examine the cultures.
Result shows that the green fluorescence was strictly confined within the yeast cells, with no detectable signal in the culture supernatant, indicating that the fusion protein was expressed but not secreted.
To evaluate the secretion efficiency of the α-factor signal peptide, we replaced the native T. versicolor laccase signal peptide with the α-signal-peptide. Following induction, we observed the cultures and measured the fluorescence intensity of the supernatant. Strong green fluorescence was observed in the culture supernatant, and its intensity increased significantly over time, indicating that the α-signal-peptide efficiently mediates EGFP secretion.
Previously, the large laccase-EGFP fusion protein failed to be secreted in Pichia pastoris. To investigate whether the EGFP tag caused steric hindrance, we constructed a new plasmid expressing only the α-signal-peptide and laccase. After induction, we collected the culture supernatants and performed a standard laccase activity assay using ABTS as the substrate. The activity of the α-Laccase (31.97 U/L) was substantially higher than that of the α-Laccase-EGFP fusion protein (15.29 U/L) and the wild-type control (11.12 U/L), confirming that removing the EGFP tag significantly enhanced both the secretion efficiency and catalytic activity of the laccase.In addition, the ability of Pichia pastoris to degrade lignin was also significantly enhanced.
After coculturing P. pastoris with genetically modified acid-tolerant KT 2440 for 1 day, we took the coculture mixture, screened for viable bacterial cells with optic microscope, and observed no living bacteria. This is probably due to P. pastoris’s inherent bactericidal ability, which makes it impossible for P. pastoris to coexist with certain types of bacteria, such as our genetically modified acid-tolerant KT 2440.
We harvested the sediments of the samples using centrifugation, weighed the sediments and plotted their mass against time before their harvest (being 12 hrs, 24 hrs, 36 hrs, 48 hrs, 60 hrs and 72 hrs respectively). Result shows that the masses of the cellulose sediments decreased as a function of time, suggesting that Trichoderma reesei TU6 is steadily degrading cellulose.
When we incubated the supernatant derived from the cultivation of genetically engineered T. reesei TU6 with lignin, a clear trend emerged. Over the course of the culture period, there was a marked decline in the lignin content, strongly suggesting that the engineered strain was having an impact on the lignin.
After 3 days of culturing, a comparison between the treated group (exposed to the supernatant from the engineered T. reesei TU6) and the untreated control group was quite revealing. The degradation rate of lignin in the treated group hit 30.1%, a figure that starkly contrasts with the control. This significant difference serves as compelling evidence that the genetically engineered T. reesei TU6 is highly effective at degrading lignin.
In addition to the lignin degradation tests, we also turned our attention to the laccase activity in the supernatants. The measurement results were striking. In the control group, the laccase activity in the supernatant was recorded at 29.19 U/L. In sharp contrast, the treated group showed a laccase activity of 79.45 U/L. This substantial increase in laccase activity in the treated group clearly points to a significant rise in the active components within the supernatant of the genetically engineered T. reesei TU6. It’s reasonable to infer that these increased active components, likely including laccase, are closely related to the enhanced lignin - degrading ability of the engineered strain.
We measured the weights of remaining vinasse after cultivating them with genetically engineered T. reesei TU6 for 3 days, and found that the mass of vinasse was reduced by half after cultivation, demonstrating the robustness of our engineered organism.
After coculturing T. reesei TU6 with genetically modified KT 2440 for 3 days, we took the coculture mixture and screened for viable bacterial cells with optic microscope at varying depths of the mixture.
On the surface of the mixture, few viable bacterial cells could be identified; but as the depth of the samples increased, viable, moving bacterial cells began to appear. And as the depth of the sample reached the 1mL scale, coexistence of T. reesei TU6 and genetically modified KT 2440 could be observed. We speculate that the bacteria might be trying to utilize the breakdown products of cellulose that are generated by T. reesei TU6 (a possible explanation for why KT 2440 could be found in close proximity with T. reesei TU6).
We selected the UTR sequence of the EGFP gene in pUC19-EGFP as the target sequence for optimization. Five optimized UTR sequences were screened, and plasmids containing these optimized sequences were obtained through full gene synthesis. Subsequently, the plasmids were transformed into E. coli BL21. After cultivation, the fluorescence intensity (excitation at 485 nm, emission at 520 nm) and OD600 values were measured for each group. The expression levels were compared using the Fluorescence/OD600 ratio. Among the five optimized UTR sequences, One (8600) showed increased expression (p=0.05688) compared to the original plasmid, indicating that this optimized UTR sequence enhanced the expression of the EGFP protein.
