Catalog
We ultimately selected Aureobasidium melanogenum BZ-11 as our chassis strain. During the cultivation of the strain, certain characteristics of BZ-11 demonstrated its potential to serve as a chassis strain for the synthetic β-glucan pathway, while other traits also presented challenges for its utilization.
Colonies of the Aureobasidium melanogenum BZ-11 strain were cultivated on both YPD (Yeast Peptone Dextrose) and PDA (Potato Dextrose Agar) media. The colony structure and microscopic observation are depicted in the figures below.
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Figure 1: Colony Morphology and Microscopic Observations of BZ-11. (a) Colony morphology of BZ-11 on YPD medium. (b) Colony morphology of BZ-11 on PDA medium. (c) Microscopic observation of BZ-11. |
Upon examination of the images, it is evident that BZ-11 secretes a substantial amount of extracellular polysaccharides when cultured on YPD medium, classifying it as a high-yielding strain for extracellular polysaccharide production. After cultivation on PDA plates, a pronounced brown-yellow halo at the colony's edge is observed, leading to the inference that BZ-11 is capable of secreting melanin extracellularly. This hypothesis was subsequently confirmed by a review of the relevant literature, which showed that Aureobasidium melanogenum not only produces melanin, but has even been used to synthesise it efficiently[1][2]. The incorporation of melanin into the extracellular polysaccharides poses a challenge for separation, thus becoming a limiting factor for the BZ-11 strain. Microscopic examination revealed the morphological characteristics and reproductive modes of individual cells.
The type strain 110.374 of Aureobasidium spp. and the strain BZ-11 were cultivated at the same situation. At the same time, the extracellular β-glucan, extracellular polymeric substances (EPS), cell wall β-glucan, and biomass titer of both strains were measured. The results are depicted in the figure below.
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Figure 2: Comparative Analysis of Strains 110.374 and BZ-11. |
Upon comparison of the biomass titers of the two strains, no significant differences were observed, indicating that their growth conditions and metabolic activities are fundamentally similar. This implies that the differences in biomass have a negligible impact on the experimental conclusions.
Under these premises, the EPS titer of BZ-11 was found to be significantly higher than that of 110.374, suggesting that BZ-11 possesses a stronger capacity for extracellular polysaccharide synthesis under the same conditions. The cell wall β-glucan content of both strains is similar, but BZ-11's EPS contains β-glucan similar in amount to that in the cell wall, whereas 110.374 does not secrete extracellular β-glucan. This indicates that BZ-11 has an inherent advantage in β-glucan production. Moreover, the extraction of extracellular β-glucan is simpler, more environmentally friendly, and BZ-11 has a clear potential for β-glucan production.
It is worth noting that the yield of extracellular polysaccharides in BZ-11 is greater than the content of extracellular β-glucan, indicating that the extracellular polysaccharides contain other types of polysaccharides.
Through the study of the manuscript, we observed that the fermentation process of Aureobasidium spp. often accompanies the production of pullulan[3]. Therefore, an enzymatic hydrolysis method was employed to detect the pullulan in the extracellular polysaccharides of the BZ-11 strain. Utilizing pullulanase, pullulan can be decomposed into maltotriose.
High-performance liquid chromatography (HPLC) analysis was conducted on maltotriose standards, pullulan standards, the supernatant of polysaccharide fermentation from the BZ-11 strain, and β-glucan standards, with the results depicted in the figure below.
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Figure 3: HPLC profiles of pullulanase hydrolysates of pullulan standard, glucan standard, exopolysaccharides produced by BZ-11, as well as maltotriose standard without pullulanase hydrolysates. |
Analysis of the maltotriose standard revealed a peak at approximately 9 minutes, which corresponds to maltotriose. Under these conditions, the enzymatic hydrolysis of pullulan standard also yielded a peak at the same retention time, indicating that the pullulanase used in the experiment is active and effective. The β-glucan standard solution did not exhibit a peak after enzymatic hydrolysis, suggesting that the presence of β-glucan does not generate maltotriose that could interfere with the experimental results. Consequently, the appearance of a maltotriose peak in the enzymatic hydrolysate of the BZ-11 strain's polysaccharide fermentation supernatant indicates that, in addition to β-glucan, the extracellular polysaccharides also contain pullulan.
In order to ensure the purity of β-glucan and to reduce the difficulty of extraction, we attempted to knock out the genes encoding key proteins in the melanin and pullulan synthesis pathways of the BZ-11 strain. Based on the genomic information of the BZ-11 strain and the modeling analysis of the dry laboratory group, the polyketide synthase gene PKS, which is crucial for melanin synthesis, and the pullulan synthesis key genes AGS2-1 and AGS2-2, were ultimately identified for knockout.
We designed a gene knockout strategy using homologous recombination to disrupt the synthesis pathways of impurities. Consequently, we amplified the FL4A-NAT-loxp plasmid along with the 3' and 5' homologous arms of the target gene to construct the gene knockout vector. The primer sequences designed for amplifying the homologous arms are presented in the table below.
In PDA medium, streak cultivation was performed for both the BZ-11 and BZW strains, with the results illustrated in the figure below.
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Figure 7: Colony morphology of BZ-11 and BZW strains. (a) Colony characteristics of the BZWΔags2-1/2 strain after cultivation; (b) Extracellular polysaccharides produced by the collected BZWΔags2-1/2 strain; (c) Colony characteristics of the BZ-11 strain after cultivation; (d) Extracellular polysaccharides produced by the collected BZ-11 strain. |
After the PKS gene knockout, melanin synthesis was completely blocked, resulting in a significant change in the color of the fermentation liquid and extracellular polysaccharides. The original strain A. melanogenum BZ-11 cultivated on PDA medium exhibited black colonies (Figure 7c and Figure 7d), whereas the A. melanogenum BZW strain showed white colony morphology without melanin production (Figure 7a and Figure 7b).
High-performance liquid chromatography (HPLC) analysis was conducted on the enzymatic hydrolysates of the exopolysaccharides from the fermentation supernatant of BZWΔags2-1/2, and the results are depicted in Figure 8.
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Figure 8: HPLC profiles of pullulanase hydrolysates of pullulan standard, glucan standard, and exopolysaccharides produced by BZ-11 and BZWΔags2-1/2, as well as the maltotriose standard without pullulanase hydrolysates. |
Using the fermentation supernatant of the original strain BZ-11, pullulan standard, β-glucan standard, and maltotriose standard as controls for comparative analysis, the results are as shown in the figure. The enzymatic hydrolysate of the exopolysaccharides from the fermentation supernatant of BZWΔags2-1/2 lacks a maltotriose peak, whereas the enzymatic hydrolysate from BZ-11 exhibits a maltotriose peak. Since pullulanase can hydrolyze pullulan to produce maltotriose but cannot hydrolyze β-glucan, this indicates that the BZWΔags2-1/2 strain is no longer capable of synthesizing pullulan.
In addition to the aforementioned analyses, Fourier-transform infrared spectroscopy (FTIR) was also conducted on the extracellular β-glucan, cell wall β-glucan of BZWΔags2-1/2, and extracellular β-glucan of BZ-11. The detection was performed using a Nicolet Nexus 470 FTIR spectrometer with a scanning range of 4000-400 cm⁻¹, a resolution of 2 cm⁻¹, and 16 scans, and the results are depicted in Figure 9.
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Figure 9: FTIR spectra of the exopolysaccharides β-glucan, cell wall β-glucan produced by BZWΔags2-1/2, and cell wall β-glucan produced by BZ-11. |
According to the FTIR analysis, all three types of polysaccharides exhibited a broad and intense O-H stretching vibration absorption peak near 3321 cm⁻¹, indicating their typical saccharide characteristics. Additionally, the C-H stretching vibration absorption peak was observed at 2919 cm⁻¹. It is also noteworthy that the extracellular polysaccharides of the original BZ-11 strain showed characteristic absorption peaks for α-configuration at 850 cm⁻¹ and β-configuration at 890 cm⁻¹, whereas the extracellular β-glucan and cell wall β-glucan of the engineered BZWΔags2-1/2 strain only exhibited a β-configuration absorption peak at 890 cm⁻¹. This suggests that the extracellular and cell wall β-glucan samples of the BZWΔags2-1/2 strain are free from contamination with α-configuration glycosidic bond polysaccharides, while the extracellular polysaccharides of the original BZ-11 strain contain both β-glucan and α-configuration glycosidic bond polysaccharides. As pullulan is an α-configuration glycosidic bond polysaccharide, this result is consistent with the high-performance liquid chromatography (HPLC) analysis, further confirming that the engineered strain no longer synthesizes pullulan.
To ensure the authenticity and reliability of the results, we also conducted 1H-13C HSQC (heteronuclear single quantum coherence) analysis using a JEOL JNM-ECP 600M HZ NMR spectrometer, and the results are shown in Figure 10.
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Figure 10: 1H-13C HSQC spectrum of exopolysaccharides β-glucan produced by BZWΔags2-1/2. |
As can be seen from the figure, the terminal carbon signal appears at 103 ppm, and the terminal hydrogen signal appears at a chemical shift of 4.5 ppm, which allows us to identify the extracellular polysaccharide as β-glucan. Additionally, the peaks at 86.84 ppm, 77.38 ppm, 73.34 ppm, 70.84 ppm, and 61.44 ppm correspond to the positions of carbons C3, C5, C2, C4, and C6, respectively. This further confirms that the extracellular polysaccharide produced by the engineered BZWΔags2-1/2 strain is β-glucan.
The consistency of the results from HPLC, infrared spectroscopy, and NMR spectroscopy indicates that after the knockout of the AGS2-1 and AGS2-1 genes, the extracellular polysaccharide product of BZWΔags2-1/2 no longer contains pullulan, and is a pure β-glucan.
To further increase the β-glucan yield of BZWΔags2-1/2, the medium was optimised.
Through optimization of the nitrogen source, the results are shown in Figure 11.
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Figure 11: Optimization of the nitrogen source in the culture medium. |
When NaNO₃ was used as the nitrogen source, the extracellular β-glucan yield of the engineered BZWΔags2-1/2 strain was significantly higher than that in the medium with (NH₄)₂SO₄. Further optimization of NaNO₃ concentration indicated that at NaNO₃ concentrations of 0.1-0.2 g/L, the β-glucan yield was the highest, reaching 25.48-26.97 g/L. At 0.2 g/L NaNO₃, the total β-glucan yield was the highest, reaching 26.97 g/L, with cell wall β-glucan yield at 10.92 g/L and extracellular β-glucan yield at 16.05 g/L.
The concentration of the carbon source (glucose) was optimized in a gradient, and the results are shown in Figure 12.
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Figure 12: Optimization of the carbon source in the culture medium. |
When the glucose concentration was within the range of 90-140 g/L, the β-glucan yield was 22.11-29.64 g/L. At a glucose concentration of 130 g/L, the β-glucan yield of the engineered BZWΔags2-1/2 strain was the highest, reaching 29.64 g/L, with cell wall β-glucan yield at 11.92 g/L and extracellular β-glucan yield at 17.81 g/L.
The β-glucan yield of the engineered BZWΔags2-1/2 strain after optimization in this experiment has significantly increased, reaching a maximum of 29.64 g/L, which is much higher than the 9.9 g/L in existing technologies. In addition, the engineered strain completely eliminated the production of the byproduct pullulan, demonstrating significant technological progress.
The final optimized culture medium composition is: 90-140 g/L glucose, 0.2 g/L yeast extract, 0.1-0.2 g/L NaNO₃, 7 g/L potassium dihydrogen phosphate, 2.5 g/L sodium phosphate dodecahydrate, 2.5 g/L magnesium sulfate heptahydrate, 0.15 g/L ferric chloride, 0.15 g/L calcium chloride, 0.02 g/L manganese sulfate, and 0.02 g/L zinc sulfate.
Inoculating the engineered BZWΔags2-1/2 strain into a fermenter containing fermentation medium for scaled-up culture and fermentation, continuous monitoring of extracellular glucan, cell wall glucan, cell dry weight, and residual glucose in the fermenter was conducted. The data recorded up to 120 hours were plotted into a line graph, as shown in Figure 13.
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Figure 13: Monitoring of extracellular glucan, cell wall glucan, cell dry weight, and residual glucose in the fermenter. |
Upon calculation, it was found that by the 120th hour, the glucose in the medium was essentially completely consumed. At this point, the total content of β-glucan, as well as the contents in the cell wall and extracellularly, all reached their peak values, with slow growth in cell dry weight.
The engineered BZWΔags2-1/2 strain was inoculated into a fermenter containing fermentation medium and fermented at 28℃-30℃. After fermentation, centrifugation was performed, and the results are shown in Figure 14.
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Figure 14: Separation and extraction of extracellular and cell wall β-glucan. |
Extracellular β-glucan was separated from the supernatant, and cell wall β-glucan was separated from the biomass. The method for separating extracellular β-glucan from the fermentation liquid was precipitation with alcohol, and the method for separating cell wall β-glucan from the microbial cells was alkali extraction. After freeze-drying, high-quality β-glucan products were obtained.
After reviewing the literature and the work of the dry laboratory group, we ultimately identified three candidate anchor proteins, AM.CWP, AM.SED, and AM.TIP, to assist in the binding of VHb to the cell wall. To compare these three options, we fused them with GFP and constructed the verification plasmids pNATX13-loxp-AM.CWP-EGFP, pNATX13-loxp-AM.SED-EGFP, and pNATX13-loxp-AM.TIP-EGFP, as shown in the figure below.
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Figure 15: pNATX13-loxp-AM.CWP-EGFP |
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Figure 16: pNATX13-loxp-AM.TIP-EGFP |
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Figure 17: pNATX13-loxp-AM.SED-EGFP |
After transformation, fluorescence observation was conducted, with results as shown in Figure 18.
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Figure 18: Fluorescence observation of AM.SED, AM.CWP, and AM.Tip as anchor proteins at 48h and 72h |
The fluorescence intensity of all three groups of experiments was enhanced along with the extension of incubation time, but the overall fluorescence intensity was not very high. It was observed that the fluorescence intensity and distribution of fluorescence with AM.CWP was significantly better than that with AM.SED and AM.Tip, and the difference in fluorescence intensity was seen at the cell wall. No significant difference in fluorescence intensity was seen at the cell wall with AM.SED and AM.Tip. Therefore, we also conducted further tests on AM.CWP and the results are shown in Figure 19.
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Figure 19: Fluorescence observation of CWP-EGFP. (A) Bright-field observation at 24h; (B) Observation under 488nm excitation light at 24h; (C) Bright-field observation at 48h; (D) Observation under 488nm excitation light at 48h; (E) Bright-field observation at 72h; (F) Observation under 488nm excitation light at 72h |
The transformed strain 48H exhibited a green fluorescent halo around the cells, which expanded with the extension of cultivation time. By the 72-hour mark, the cells were uniformly green, and it was observable that the fluorescence intensity was not high. Moreover, the difference in fluorescence intensity between inside the cells and on the cell wall was not significant. This observation suggests that the fusion protein constructed could potentially assist VHb in binding to the cell wall of BZWΔags2-1/2, thereby providing the cells with a more oxygen-rich microenvironment and enhancing the production of glucan. But our work still needs to be further optimised.
A region rich in Ser/Thr structural domain is located near the C-terminal anchoring sequence, and this area contains multiple O-glycosylation sites. The serine or threonine at these sites may covalently bind to glucans on the cell wall. These sites could be crucial for the stable attachment of the anchor protein to the cell wall. In the initial experiments, we did not include this sequence in the design of the fusion protein. This might be one of the reasons for the low fluorescence intensity and the lack of significant difference in fluorescence intensity between the cell wall and the inside of the cell. The next experimental approach is to incorporate the aforementioned sequence into the design and optimize the length of the sequence included.
Firstly, adjust the C-terminal domain of the anchor protein until satisfactory results are achieved, characterized by significantly higher fluorescence intensity on the cell wall compared to the interior of the cell, and a high overall fluorescence intensity. Then, construct an expression vector for VHb using the optimal anchor protein sequence. After transforming the plasmid into BZWΔags2-1/2, perform PCR validation to confirm the insertion of the VHb gene into the genome. Subsequently, use Cre recombinase to eliminate the resistance gene. Following this, conduct CO-difference spectrum and oxygen uptake rate (OUR) analysis to verify whether there is an enhancement in the oxygen uptake capacity of the strain. In addition, we can employ real-time quantitative PCR technology to measure the transcription levels of genes involved in the β-glucan synthesis pathway, to assess the impact of VHb on β-glucan synthesis. Finally, we can also construct a fusion protein of VHb-EGFP to directly observe the expression and distribution of VHb [5].
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