1 Overview
Our project primarily consists of the red algal polysaccharide utilization module and the Rh1 synthesis module. This page presents the experimental results of our project and our explanation of the data. It mainly includes plasmid construction maps for each module, screening results of engineered strains, determination of fermentation conditions and product yields.
2 Utilization of Red Algal Polysaccharides
2.1 Hydrolase Plasmid Construction
We selected three agarases and two neoagarobiose hydrolases from different sources. After codon optimization, six plasmids were constructed through combinatorial assembly. The p426 Gal backbone vector was used to construct Saccharomyces cerevisiae expression plasmids[1], featuring URA3 as the selectable marker, ampicillin (Amp) resistance, and the strong promoters Pgal7 and Pgal1. Secretion was directed using the $\alpha$-factor hydrolase signal peptide (BBa_258RYIFY).
Maps of the 6 constructed plasmids
Target fragments were successfully amplified and assembled into the vector using Gibson Assembly. Constructed plasmids were transformed into E. coli DH5 $\alpha$. Successful ligation was confirmed by colony PCR and sequencing, yielding agarase system expression vectors.
2.2 Screening Engineered Strains
The six plasmids were transformed into wild-type S. cerevisiae CEN.PK2-1D using the lithium acetate method, yielding six engineered strains (Sq-Ag1, Sq-Ag2, Sq-Ag3, Sq-Ag4, Sq-Ag5, Sq-Ag6).
Genotypes of engineered strains
| Strain | Genotype | |
|---|---|---|
| Agarase | Neoagarobiose Hydrolase | |
| Sq-Ag1 |
Aga3463 |
NH852 |
| Sq-Ag2 |
AqAga |
NH852 |
| Sq-Ag3 |
PdAgaC |
NH852 |
| Sq-Ag4 |
Aga3463 |
agaNash |
| Sq-Ag5 |
AqAga |
agaNash |
| Sq-Ag6 |
PdAgaC |
agaNash |
| Sq-pls |
p426 empty plasmid |
|
| WT |
The control group without any treatment |
|
All six strains were cultured in uracil-deficient YPD medium. Qualitative detection using Lugol's iodine plate assay confirmed extracellular agarase secretion capability for all strains.
As can be seen from the size of the transparent zones, Sq-Ag4,Sq-Ag5, and Sq-Ag6 exhibit strong secretion abilities.
Results of Lugol's iodine plate assay
Activated seed cultures of the six engineered strains were transferred to YPD medium for agarase and neoagarobiose hydrolase activity assays.
By constructing a standard curve of enzyme activity and comparing it with the samples, we obtained the enzyme activity data of the engineering strain.[2]
a) Galactose standard curve for neoagarobiose hydrolase activity.
b) Galactose standard curve for agarase activity.
c) Enzyme activity assays for agarase and neoagarobiose hydrolase.
By combining the results of the Lugol's iodine solution plate assay and the enzyme activity assay, the optimal enzyme combination was the agarase gene AqAga (BBa_258028F8)from Aquimarina agarilytica ZC1 and the neoagarobiose hydrolase gene agaNash (BBa_257L75AN) from Cellvibrio sp. OA-2007. This combination (strain Sq-Ag5) was selected for subsequent engineering.
2.3 Optimize the MVA pathway
Construction of Cas9-sgRNA Plasmid
sgRNA 20 nt sequences were selected using the CHOPCHOP website and were cloned into a Cas9 expression plasmid, constructing individual GAL80-targeting plasmids ($p426-P_{TEF1}-SpCas9-T_{CYC1}-P_{SNR52}-sgRNA-T_{SUP4}$). Each plasmid expresses Cas9 protein and a specific sgRNA in yeast to form a complex for precise genomic cutting.
Plasmid map
Gel electrophoresis of the PCR-linearized $p426-P_{TEF1}-SpCas9-T_{CYC1}-P_{SNR52}-sgRNA-T_{SUP4}$ plasmid (10251 bp fragment)
Construction of Donor DNA
Homology arms for the GAL80 locus were introduced via primers for tHMG1 and IDI1. Fragments were amplified by PCR and assembled.
Linear donor DNA map
The assembled donor was transformed into E. coli DH5 $\alpha$. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was PCR-amplified, and the donor DNA fragment was purified using a SanPrep PCR Product Purification Kit.
Successfully transformed E. coli DH5 $\alpha$ colonies
Gel electrophoresis confirming successful vector ligation
Screening Engineered Strains
Plasmids were transformed into prepared competent S. cerevisiae CEN.PK2-1D. Primary and counter-selection were performed to obtain strains with successful genomic integration. Primary screening was performed using colony PCR.
Primary screening was performed using colony PCR.
Primary selection plate results
Gel electrophoresis results showing bands of expected length, indicating successful genomic integration of the target gene
Counter-selection was used to remove the URA3 selectable marker from positive clones.
Counter-selection plate results
Gel electrophoresis results confirming successful excision of the URA3 marker
The final engineered strain, S. cerevisiae Sq-0, with successful genomic integration and marker excision, was obtained.
Squalene content detection showed that overexpressing tHMG1 and IDI1 to optimize the MVA pathway significantly increased squalene production. Strain Sq-0 was used as the starting strain for subsequent engineering.
By preparing a squalene standard curve and comparing it with the samples, we obtained the squalene yield data.[3]
Squalene HPLC standard curve
HPLC chromatogram of squalene from Sq-0 fermentation broth
3 Determination of Fermentation Conditions
3.1 Liquefaction Condition Determination
YPA medium (25 mL) was treated with HCl at final concentrations ranging from 0.001 M to 0.01 M at 121°C for 20 min.
The results showed that an extremely low acid concentration (0.005 M hydrochloric acid) could break down the structure of agar, rendering it fluid. Moreover, it remained non-coagulated throughout the yeast shake flask culture and gradually became clear, indicating that the agar had been completely utilized.
State of the medium during fermentation
HPLC detection confirmed that 0.005 M HCl liquefaction did not degrade agar into neoagarobiose or monosaccharides. The medium pH was adjusted to 6.0 for yeast fermentation experiments.
HPLC chromatogram of yeast medium liquefied with 0.005 M HCl
3.2 Carbon Source Condition Determination
Plasmid p426-AqAga-agaNash was transformed into engineered strain Sq-0 using the lithium acetate method, yielding engineered strain Sq-Ag. Orthogonal fermentation experiments were conducted in media liquefied with 0.005 M HCl.
Strain Sq-Ag successfully degraded and saccharified red algal polysaccharides stepwise. Squalene yield increased with red algal polysaccharide concentration up to ≤ 25 g/L. Higher concentrations (>25 g/L) inhibited further increases. The highest squalene yield of 667.02 mg/L was achieved with 10 g/L glucose and 25 g/L red algal polysaccharides.
Orthogonal experimental results of squalene synthesis with glucose and agar in flasks
4 Promoter Engineering
4.1 Promoter Screening
We screened 3 constitutive promoters and 3 inducible promoters, constructed plasmids with 18 promoter combinations, and transformed them into yeast cells.[4]
Yeast cells were cultured for 96 hours under the conditions of 5 g glucose and 25 g agar, and the squalene yield was detected. The inducible combination of GAL10 (agarase) + GAL7 (neoagarobiose hydrolase) was optimal, increasing the squalene yield to 594.68 mg/L.
Results of Squalene Fermentation with 18 Promoter Combinations
4.2 Insertion of Chimeric Intron
The RPS25Ai intron was inserted into the proximal regions of the Pgal10 and Pgal7 promoters.
Construct the GAL10p-RPS25Ai chimeric promoter
Yeast cells were cultured for 96 hours under the conditions of 5 g glucose and 25 g agar, and the squalene yield was detected. According to the experimental results, the highest squalene yield (682 mg) was achieved when the RPS25Ai intron was inserted into the proximal region of the Pgal10 promoter, while no chimeric intron was inserted into the Pgal7 promoter
Comparison of Squalene Fermentation Before and After Promoter Engineering Modification
The insertion of chimeric introns into promoters could enhance the expression of agarase and neoagarobiose hydrolase genes in Saccharomyces cerevisiae to varying degrees, leading to an increase in squalene yield. Through promoter engineering, the squalene yield has been significantly improved, which is more conducive to obtaining a higher yield of the rare ginsenoside Rh1 in subsequent experiments.
5 Squalene Production from Red Algal Polysaccharides
We finally used purchased red algae, which were dried and ground into powder to prepare YPDA medium for the fermentation culture of Sq - Ag. As shown in the figure, from 0 to 24 h, Saccharomyces cerevisiae consumed glucose for fermentation, resulting in a decrease in reducing sugar concentration and an increase in ethanol concentration. From 24 to 48 h, the strain began to secrete hydrolases, decomposing agar into monosaccharides, leading to an increase in reducing sugar content and a significant increase in squalene yield. From 48 h to 120 h, the bacterial growth entered the stationary phase, and the number no longer changed significantly, while squalene continued to be synthesized, indicating that the Sq - Ag strain still had strong squalene synthesis ability in the stationary phase. After five days of fermentation, the squalene yield reached 908 mg/L. The overall fermentation process showed a reasonable metabolic and product synthesis pattern, demonstrating the feasibility and efficiency of using red algal polysaccharides to ferment and produce squalene.
Squalene yield from fermentation
6 Rh1 Synthesis
The heterologous pathway enzymes for rare ginsenoside Rh1 were integrated into specific loci of wild-type S. cerevisiae CEN.PK2-1D and S. cerevisiae Sq-0.
6.1 Insert the PgDDS gene at the X-3 locus
Construction of Cas9-sgRNA Plasmid (X-3 Locus)
SgRNA 20 nt sequences were designed using CHOPCHOP and were cloned into a Cas9 expression plasmid, constructing individual targeting plasmids (p426-sgRNA-X-3).
Construction of Donor DNA (X-3 Locus - PgDDS)
Homology arms for the X-3 locus were introduced via primers for PgDDS (BBa 255LOGT6). The fragment was amplified by PCR and assembled via fusion PCR.
Linear donor DNA map
The donor was transformed into E. coli DH5$\alpha$. Positive clones were selected using ampicillin resistance. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was amplified using primers X-3-TADH1-F and X-3-Pgal1,10-R, and the donor DNA fragment was purified.
Successfully transformed E. coli DH5 $\alpha$
Gel electrophoresis confirming successful vector construction
Result Verification (X-3 Locus)
Strains were fermented for 144 h in medium containing 20 g/L glucose. Dammarenediol-II production was measured after integrating PgDDS. Dammarenediol-II yield reached 1 g/L.
a) Dammarenediol-II HPLC standard curve.
b) Dammarenediol-II production at 144 h after PgDDS integration
6.2 Insert the CYP716A47 and PgCPR1 genes at the XI -3 locus
Construction of Cas9-sgRNA Plasmid (XI-3 Locus)
sgRNA 20 nt sequences were designed using CHOPCHOP. sgRNA sequences were cloned into a Cas9 expression plasmid, constructing individual targeting plasmids (p426-sgRNA-XI-3).
Construction of Donor DNA (XI-3 Locus - CYP716A47, PgCPR1)
Homology arms for the XI-3 locus were introduced via primers forCYP716A47 and PgCPR1. Fragments were amplified by PCR and assembled via fusion PCR.
Linear donor DNA map
The donor was transformed into E. coli DH5$\alpha$. Positive clones were selected using ampicillin resistance. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was amplified using primers $XI-3-P_{gal1,10}-F and XI-3-T_{CYC1}-R$, and the donor DNA fragment was purified.
Successfully transformed E. coli DH5 $\alpha$
Gel electrophoresis confirming successful vector construction
Result Verification (XI-3 Locus)
Strains were fermented for 144 h in medium containing 20 g/L glucose. Protopanaxadiol (PPD) production was measured after integrating CYP716A47 and PgCPR1. PPD yield reached 273.8 mg/L.
a) PPD HPLC standard curve.
b) PPD production at 144 h after CYP716A47 and PgCPR1 integration
Construction of Cas9-sgRNA Plasmid (LPP1 Locus)
sgRNA 20 nt sequences were designed using CHOPCHOP. sgRNA sequences were cloned into a Cas9 expression plasmid, constructing individual targeting plasmids (p426-sgRNA-LPP1).
6.3 Insert the CYP716A53v2, UGTPg100 genes at the LPP1 Locus
Construction of Donor DNA (LPP1 Locus - CYP716A53v2, UGTPg100)
Homology arms for the LPP1 locus were introduced via primers for CYP716A53v2 and UGTPg100. Fragments were amplified by PCR and assembled via fusion PCR.
Linear donor DNA map
The donor was transformed into E. coli DH5$\alpha$. Positive clones were selected using ampicillin resistance. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was amplified using primers $LPP1-T_{ADH1}-F and LPP1-T_{ALT1}-R$, and the donor DNA fragment was purified.
Successfully transformed E. coli DH5 $\alpha$
Gel electrophoresis confirming successful vector construction
Result Verification (LPP1 Locus)
Strains were fermented for 144 h in medium containing 20 g/L glucose. Rare ginsenoside Rh1 production was measured after integrating CYP716A53v2 (BBa_25NSM6TW) and UGTPg100 (BBa_255ROLW2). Rh1 yield reached 170.8 mg/L.
a) Rh1 HPLC standard curve.
b) Rh1 production at 144 h after CYP716A53v2 and UGTPg100 integration
Screening Engineered Strains (Rh1 Pathway)
Plasmids were transformed into prepared competent S. cerevisiae CEN.PK2-1D. Primary and counter-selection were performed to obtain strains with successful genomic integration at the X-3, XI-3, and LPP1 loci.
Primary screening used colony PCR with verification primers for the X-3, XI-3, and LPP1 loci.
Primary selection plate results.
Gel electrophoresis results showing bands of expected length, indicating successful genomic integration
Counter-selection was used to remove the URA3 selectable marker from positive clones.
Counter-selection plate results
Gel electrophoresis results confirming successful excision of the URA3 marker
The final engineered strains with successful genomic integration and marker excision were obtained: S. cerevisiae SC0Rh1 (control, based on CEN.PK2-1D) and S. cerevisiae Rh1-con (based on Sq-0).
6.4 Rh1 Synthesis Using Red Algal Polysaccharides
Plasmid p426-AqAga-agaNash was transformed into engineered strains S. cerevisiae SC0Rh1 and S. cerevisiae Rh1-con using the lithium acetate method, yielding engineered strains S. cerevisiae SC0Rh1-Ag and S. cerevisiae Rh1-Ag.
Strains were fermented in YPDA medium with 10 g/L glucose + 25 g/L agar at 30°C, 220 rpm for 144 h. Samples were taken at 0, 12, 24, 48, 72, 96, 120, and 144 h to measure sugar consumption, cell growth, and Rh1 production. Results showed that compared to control strain Rh1-con, Rh1 production in Rh1-Ag significantly increased during the later fermentation stage (72-144 h), reaching a maximum yield of 141.78 mg/L at 144 h. No Rh1 was detected in S. cerevisiae SC0Rh1-Ag.
