Design
Through literature research, it was found that the agar in red algae needs to be hydrolyzed into monosaccharides in two steps. First, agarase hydrolyzes it into neoagarobiose, and then neoagarobiose hydrolase hydrolyzes it into galactose and 3,6-anhydro-L-galactose. We decided to introduce heterologous hydrolases into Saccharomyces cerevisiae to achieve extracellular secretion of hydrolases.
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Test
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CYCLE 1: Expressing Agarase and Neoagarobiose Hydrolase
Design
Through literature research, it was found that the agar in red algae needs to be hydrolyzed into monosaccharides in two steps. First, agarase hydrolyzes it into neoagarobiose, and then neoagarobiose hydrolase hydrolyzes it into galactose and 3,6-anhydro-L-galactose. We decided to introduce heterologous hydrolases into Saccharomyces cerevisiae to achieve extracellular secretion of hydrolases.
Build
We obtained the signal peptide gene from the α-factor mutant of Saccharomyces cerevisiae, which contains the Kozak sequence and helps to enhance the efficiency of translation initiation. Three types of agarases and two types of neoagarobiose hydrolases from different sources were screened, and 6 types of plasmids were constructed. We introduced the 6 types of plasmids into the Saccharomyces cerevisiae CEN.PK2-1D by lithium acetate transformation method, and obtained 6 engineered strains (namely Sq-Ag1, Sq-Ag2, Sq-Ag3, Sq-Ag4, Sq-Ag5, Sq-Ag6).
Table 1 Gene Sources of 5 Hydrolases
Table 2 Gene Sources of 5 Hydrolases
A
B
C
D
E
F
Figure Plasmids of 6 Hydrolases
Test
We cultured the 6 strains in defective medium and conducted qualitative detection using the Lugol's iodine plate method. As shown in the figure, large transparent hydrolysis circles were formed around the colonies, indicating that all 6 strains had the ability to secrete agarase extracellularly.
Lugol's iodine staining result diagram
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This cycle initially verified the ability of the reconstructed yeast to decompose agar, but we need to further conduct qualitative research on the activity of hydrolases to obtain the combination with the best activity.
CYCLE 2: Expressing Agarase and Neoagarobiose Hydrolase
Design
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CYCLE 3: Expressing Agarase and Neoagarobiose Hydrolase
Design
After obtaining the Sq-Ag5 strain with the highest enzyme activity, we hoped that Sq-Ag5 could integrate the exogenous enzymatic hydrolysis pathway and the endogenous fermentation pathway, and successfully produce the rare ginsenoside precursor squalene using agar. We first attempted to conduct fermentation experiments using 20 g/L glucose and 20 g/L agar.
Build
We inoculated the Sq-Ag5 strain on a nutrient-deficient plate, then carried out shake flask fermentation in YPD medium and YPA liquid medium respectively, and finally detected the squalene content in the fermentation broth using a HPLC.
Test
Since the fermentation temperature of yeast is 30°C, the medium with only agar added solidified, making it impossible to detect the product. Strangely, the group with only glucose added also had a very low squalene content, only 5.89 mg/L, which could not provide sufficient precursor substances for the subsequent conversion of ginsenosides.
Figure HPLC Detection Chromatogram of Sq-Ag5 Squalene Fermentation Broth
Learn
After communication with Dr. Xie, it was initially determined that the MVA pathway for squalene production in Saccharomyces cerevisiae was limited. Literature research showed that up-regulating the expression of genes encoding key enzymes in the MVA pathway can effectively increase the overall flux of this metabolic pathway.
CYCLE 4: Optimization of the MVA Pathway
CYCLE 4.1: Try to Produce Squalene Using Sq-Ag5
Design
After obtaining the Sq-Ag5 strain with the highest enzyme activity, we hoped that Sq-Ag5 could integrate the exogenous enzymatic hydrolysis pathway and the endogenous fermentation pathway, and successfully produce the rare ginsenoside precursor squalene using agar. We first attempted to conduct fermentation experiments using 20 g/L glucose and 20 g/L agar.
Build
We inoculated the Sq-Ag5 strain on a nutrient-deficient plate, then carried out shake flask fermentation in YPD medium and YPA liquid medium respectively, and finally detected the squalene content in the fermentation broth using a HPLC.
Test
Since the fermentation temperature of yeast is 30°C, the medium with only agar added solidified, making it impossible to detect the product. Strangely, the group with only glucose added also had a very low squalene content, only 5.89 mg/L, which could not provide sufficient precursor substances for the subsequent conversion of ginsenosides.
Figure HPLC Detection Chromatogram of Sq-Ag5 Squalene Fermentation Broth
Learn
After communication with Dr. Xie, it was initially determined that the MVA pathway for squalene production in Saccharomyces cerevisiae was limited. Literature research showed that up-regulating the expression of genes encoding key enzymes in the MVA pathway can effectively increase the overall flux of this metabolic pathway.
CYCLE 4: Optimization of the MVA Pathway
CYCLE 4.2: Try to Produce Squalene Using Sq-Ag5
Design
According to the conclusion of CYCLE 4.1, we found that the excessively high concentration of red algae polysaccharides would increase the viscosity of the medium, which is unfavorable for the growth of strains. Therefore, it is important to explore the specific conditions for the liquefaction under the action of low-concentration hydrochloric acid: it is necessary to ensure the fluidity of the fermentation medium to facilitate the increase of microbial movement; it is also necessary to ensure that no monosaccharides are hydrolyzed during the liquefaction pretreatment.
Build
We used hydrochloric acid with a final concentration gradient of 0.001 to 0.01 M in YPA medium and carried out high-temperature treatment. To verify whether the polysaccharides were hydrolyzed during the liquefaction process, we planned to use the engineered strain Sq-Ag5 for fermentation experiments after finding the optimal liquefaction conditions to detect whether neoagarobiose and galactose were produced.
Test
The results showed that an extremely low acid concentration (0.005 M hydrochloric acid) could open the structure of agar to be in a fluid state, and the medium did not solidify during the entire shake flask culture of yeast. After liquefying the yeast medium with 0.005 M hydrochloric acid, no neoagarobiose or monosaccharides were detected by HPLC, and the medium could be used for yeast fermentation experiments after adjusting the pH to 6.0.
Figure Medium State During Fermentation
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During the fermentation process, we monitored the state of the medium at different stages (0 h, 48 h, 120 h) and observed the effect of hydrochloric acid concentration on the fluidity of the medium. The above phenomena confirmed the effectiveness of this experimental system.
CYCLE 5: Fermentation Production of Squalene by the Engineered Strain Sq-Ag Using Red Algae Polysaccharides as Substrate
CYCLE 5.1: Determine Liquefaction Conditions
Design
There is a distribution limit of metabolic resources in Saccharomyces cerevisiae, so it is necessary to establish a dynamic balance between cell growth and target product synthesis. In the early stage of the experiment, it was predicted by the Flux Balance Analysis (FBA) model that the flux of the Rh1 synthesis reaction is greater under the condition of mixed carbon sources. To optimize the carbon source utilization efficiency, we designed an orthogonal fermentation experiment to explore the optimal ratio of glucose to red algae polysaccharides. The strategy was to use low-concentration glucose to initiate microbial growth, while maximizing the efficiency of galactose as a carbon source.
Build
We introduced the plasmid p426-AqAga-agaNash into the engineered strain Sq-0 by lithium acetate transformation method to obtain the engineered strain Sq-Ag, and carried out orthogonal fermentation experiments in a series of media (liquefied with 0.005 M hydrochloric acid).
Test
Through detection, it was found that yeast could achieve the step-by-step degradation and saccharification of agar from scratch. The experimental results showed that 10 g/L glucose and 25 g/L agar could achieve the highest squalene yield of 667.02 mg/L.
Figure Orthogonal Experimental Results of Squalene Synthesis by Glucose and Agar in Shake Flasks
Learn
When the concentration of red algae polysaccharides was ≤25 g/L, the squalene yield increased with the increase of red algae polysaccharide concentration. However, high-concentration polysaccharides (exceeding 25 g/L) began to inhibit the increase of squalene. The possible reason is that high-concentration red algae polysaccharides lead to excessively high medium viscosity, which affects oxygen transfer or cell metabolism. This reminds us that in the development of new carbon source fermentation processes, the selection of carbon sources requires multi-dimensional systematic evaluation.
CYCLE 5: Fermentation Production of Squalene by the Engineered Strain Sq-Ag Using Red Algae Polysaccharides as Substrate
CYCLE 5.2: Orthogonal Experiment