Endophytes, biotransforming microorganisms, and engineering microbial factories for triterpenoid saponins production

Yao, Lu; Wang, Juan; He, Jin; Huang, Luqi; Gao, Wenyuan

doi:10.1080/07388551.2020.1869691

Cited by 12 publications

(5 citation statements)

References 165 publications

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“…S. cerevisiae is an ideal platform for heterologous biosynthesis of triterpenoids [ 31 ]. However, the low catalytic efficiency of cytochrome P450 enzymes (P450s), which require NADPH-cytochrome P450 reductases (CPR) to provide electrons, was the primary challenge for terpenoids synthesis [ 32 ].…”

Section: Resultsmentioning

confidence: 99%

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

Zhu

Peng

et al. 2022

Microb Cell Fact

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Background Ginsenosides are Panax plant-derived triterpenoid with wide applications in cardiovascular protection and immunity-boosting. However, the saponins content of Panax plants is fairly low, making it time-consuming and unsustainable by direct extraction. Protopanaxadiol (PPD) is a common precursor of dammarane-type saponins, and its sufficient supply is necessary for the efficient synthesis of ginsenoside. Results In this study, a combinational strategy was used for the construction of an efficient yeast cell factory for PPD production. Firstly, a PPD-producing strain was successfully constructed by modular engineering in Saccharomyces cerevisiae BY4742 at the multi-copy sites. Then, the INO2 gene, encoding a transcriptional activator of the phospholipid biosynthesis, was fine-tuned to promote the endoplasmic reticulum (ER) proliferation and improve the catalytic efficiency of ER-localized enzymes. To increase the metabolic flux of PPD, dynamic control, based on a carbon-source regulated promoter PHXT1, was introduced to repress the competition of sterols. Furthermore, the global transcription factor UPC2-1 was introduced to sterol homeostasis and up-regulate the MVA pathway, and the resulting strain BY-V achieved a PPD production of 78.13 ± 0.38 mg/g DCW (563.60 ± 1.65 mg/L). Finally, sugarcane molasses was used as an inexpensive substrate for the first time in PPD synthesis. The PPD titers reached 1.55 ± 0.02 and 15.88 ± 0.65 g/L in shake flasks and a 5-L bioreactor, respectively. To the best of our knowledge, these results were new records on PPD production. Conclusion The high-level of PPD production in this study and the successful comprehensive utilization of low-cost carbon source -sugarcane molassesindicate that the constructed yeast cell factory is an excellent candidate strain for the production of high-value-added PPD and its derivativeswith great industrial potential. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

Zhu

Peng

et al. 2022

Microb Cell Fact

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“…Several methods for ginsenoside production are being sourced to meet this demand. Presently, the methods employed for CK production include microbial engineering, biotransformation, enzymatic and chemical processes 57 , 62 – 64 . The latter two processes are deemed expensive and low-yielding, bringing the focus on the former two with a preference for biotransformation.…”

Section: Discussionmentioning

confidence: 99%

Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation

Rai,

Singh,

Shaanker

et al. 2024

Sci Rep

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Endophytes of Panax have the potential to produce their host plant secondary metabolites, ginsenosides. Panax sokpayensis, an endemic traditional medicinal plant of the Sikkim Himalayas was explored for the isolation of endophytic fungi. In the present study, we have isolated 35 endophytic fungal cultures from the rhizome of P. sokpayensis and screened for ginsenosides production by HPLC by comparing the peak retention time with that of standard ginsenosides. The HPLC analysis revealed that out of 35 isolates, the mycelial extracts of four fungal endophytes (PSRF52, PSRF53, PSRF49 and PSRF58) exhibited peaks with a similar retention time of the standard ginsenoside, Compound K (CK). LC–ESI–MS/MS analysis led to the confirmation of ginsenoside CK production by the four fungal endophytes which showed a compound with m/z 639.6278, similar to that of standard ginsenoside CK with yield in potato dextrose broth flask fermentation ranging from 0.0019 to 0.0386 mg/g of mycelial mass in dry weight basis. The four prospective fungal endophyte isolates were identified as Thermothielavioides terrestris PSRF52, Aspergillus sp. PSRF49, Rutstroemiaceae sp. strain PSRF53, and Phaeosphaeriaceae sp. strain PSRF58 based on ITS sequencing. The present finding highlights the need for further study on growth optimization and other culture parameters to exploit the endophytes as an alternative source for ginsenoside CK production.

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“…Significant progress in triterpenoid production has recently been reported using microorganisms, particularly S. cerevisiae , as alternative hosts (for a review, see Yao et al, 2020 ). However, when using such microbial systems, extensive metabolic engineering to maximize upstream carbon flux flow from acetyl-CoA to 2,3-oxidosqualene and improving enzyme activities by codon optimization are generally required to achieve high-yield production.…”

Section: Discussionmentioning

confidence: 99%

High-yield bioactive triterpenoid production by heterologous expression in Nicotiana benthamiana using the Tsukuba system

et al. 2022

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Oleanolic acid is a pentacyclic triterpenoid found in numerous plant species and is a precursor to several bioactive triterpenoids with commercial potential. However, oleanolic acid accumulates at low levels in plants, and its chemical synthesis is challenging. Here, we established a method for producing oleanolic acid in substantial quantities via heterologous expression of pathway enzymes in Nicotiana benthamiana. The “Tsukuba system” is one of the most efficient agroinfiltration-based transient protein expression systems using the vector pBYR2HS, which contains geminiviral replication machinery and a double terminator for boosting expression. Additionally, the pBYR2HS vector contains an expression cassette for the gene-silencing suppressor p19 protein from tomato bushy stunt virus, which can also contribute to enhancing the expression of target proteins. In this study, we evaluated the applicability of this system to heterologous triterpenoid production in N. benthamiana. Medicago truncatula cytochrome P450 monooxygenase (CYP) 716A12 is the first enzyme to be functionally characterized as β-amyrin C-28 oxidase producing oleanolic acid. A mutant CYP716A12 (D122Q) with improved catalytic activity engineered in our previous study was co-expressed with other enzymes in N. benthamiana leaves. Using pBYR2HS, oleanolic acid yield was increased 13.1-fold compared with that using the conventional binary vector, indicating the advantage of the Tsukuba system. We also demonstrated the efficacy of co-expressing a mutant Arabidopsis thaliana HMGR1 catalytic domain, additional NADPH-cytochrome P450 reductase (CPR) transferring electrons to heterologous CYPs, and application of ascorbic acid for preventing leaf necrosis after agroinfiltration, to improve product yield. As a result, the product yields of both simple (β-amyrin) and oxidized (oleanolic acid and maslinic acid) triterpenoids were significantly improved compared with the previously reported yield in heterologous triterpenoid production in N. benthamiana leaves.

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Endophytes, biotransforming microorganisms, and engineering microbial factories for triterpenoid saponins production

Cited by 12 publications

References 165 publications

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation

High-yield bioactive triterpenoid production by heterologous expression in Nicotiana benthamiana using the Tsukuba system

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