Overproduction of Patchoulol in Metabolically Engineered <i>Komagataella phaffii</i>

Luo, Guangjuan; Lin, Ying; Chen, Shuting; Xiao, Ruiming; Zhang, Jiaxin; Li, Cheng; Sinskey, Anthony J.; Ye, Lei; Liang, Shuli

doi:10.1021/acs.jafc.2c08228

Cited by 12 publications

(4 citation statements)

References 42 publications

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“…2A and B), potentially in uencing the differences between strains. Compared to other engineered strains of K. pha i under similar culture conditions, UA2-1 exhibited a biomass of less than 30 at 120 h, signi cantly lower than others with a biomass of 40-50 in same timeframe 32,33 . We hypothesized that the growth inhibition of UA2-1 could be attributed to the toxicity of p-benzoquinone, consequently impacting β-Arbutin titer.…”

Section: Arbutin Production Pathwaymentioning

confidence: 78%

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

yang,

Yang,

Zhao

et al. 2024

Preprint

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Background β-Arbutin, found in the leaves of bearberry, stands out as one of the globally acknowledged eco-friendly whitening additives in recent years. However, the natural abundance of β-Arbutin is low, and the cost-effectiveness of using chemical synthesis or plant extraction methods is low, which cannot meet the requirements. While modifying the β-Arbutin synthesis pathway of existing strains is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application. Results In this study, we established a biosynthetic pathway in Komagataella phaffii for β-Arbutin production with a titer of 1.58 g/L. Through diverse metabolic strategies, including fusion protein construction, enhancing shikimate pathway flux, and augmenting precursor supplies (PEP, E4P, and UDPG), we significantly increased β-Arbutin titer to 4.32 g/L. Further optimization of methanol concentration in shake flasks led to a titer of 6.32 g/L titer after 120 h of fermentation, representing a four-fold increase over the initial titer. In fed-batch fermentation, strain UA3-10 set a record with the highest production to date, reaching 128.6 g/L in a 5 L fermenter. Conclusions This is the highest yield in the fermentation tank level of using microbial cell factories for de novo synthesis of β-Arbutin. Applying combinatorial engineering strategies has significantly improved the β-Arbutin yield in K. phaffii and is a promising approach for synthesizing functional products using a microbial cell factory. This study not only advances low-cost fermentation-based production of β-Arbutin but also establishes K. phaffii as a promising chassis cell for synthesizing other aromatic amino acid metabolites.

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Section: Arbutin Production Pathwaymentioning

confidence: 78%

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

yang,

Yang,

Zhao

et al. 2024

Preprint

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“…The upper organic phase was then collected, an appropriate amount of anhydrous sodium sulfate was added, and the mixture was gently inverted several times before being left for approximately 30 min for dewatering. The upper organic phase was centrifuged at 12,000 rpm for 2 min, diluted with n-hexane, and filtered using an organic filter membrane of 0.22 µm [9,11]. Patchoulol was quantitatively determined using GC-MS (GC-2010 Plus and GCMS-QP2010, Shimadzu, Kyoto, Japan) with DB-5MS (30 m × 0.25 mm, 0.25 µm, Agilent, Santa Clara, CA, USA).…”

Section: Extraction and Detection Methodsmentioning

confidence: 99%

Metabolic Engineering for Efficient Synthesis of Patchoulol in Saccharomyces cerevisiae

Tao,

Du,

Chen

et al. 2024

Fermentation

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Patchoulol is a natural sesquiterpene alcohol with extensive applications in cosmetics and pharmaceuticals. In this study, we first constructed the synthesis pathway of patchoulol in Saccharomyces cerevisiae by expressing the patchoulol synthase PTS gene using the strong promoter GAL1. Afterward, the metabolic flux of the precursor was enhanced by strengthening the mevalonate pathway and balancing the precursor competition pathway, resulting in a 32.74-fold increase in patchoulol production. Subsequently, the supply of acetyl-CoA in yeast was increased by modifying transcriptional regulators and modulating the acetyl-CoA pathway, and the titer of patchoulol reached 155.94 mg/L. Finally, optimization of the fermentation conditions resulted in a titer of 195.96 mg/L in the shake flasks. Further, batch-fed fermentation in a 5 L bioreactor yielded 1.95 g/L. This work accelerated the development of a microbial cell factory for the production of patchoulol.

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“…Although the rapid development in genetic tools facilitates the metabolic engineering of P. pastoris , 19 − 21 engineering P. pastoris for chemicals overproduction from sole methanol remains challenging because the process of metabolizing methanol is complex and relatively unclear. In previous studies, nutrient-rich medium or additional carbon sources such as glucose are often required to increase the titer of target chemicals or to improve cell growth during methanol biotransformation, 22 , 23 which at the same time increases the production cost and the complexity of the cultivation process.…”

Section: Introductionmentioning

confidence: 99%

Engineering Yeast Peroxisomes for α-Bisabolene Production from Sole Methanol with the Aid of Proteomic Analysis

Gao,

Hou,

Cai

et al. 2024

JACS Au

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Microbial metabolic engineering provides a feasible approach to sustainably produce advanced biofuels and biochemicals from renewable feedstocks. Methanol is an ideal feedstock since it can be massively produced from CO 2 through green energy, such as solar energy. However, engineering microbes to transform methanol and overproduce chemicals is challenging. Notably, the microbial production of isoprenoids from methanol is still rarely reported. Here, we extensively engineered Pichia pastoris (syn. Komagataella phaffii) for the overproduction of sesquiterpene αbisabolene from sole methanol by optimizing the mevalonate pathway and peroxisomal compartmentalization. Furthermore, through label-free quantification (LFQ) proteomic analysis of the engineered strains, we identified the key bottlenecks in the peroxisomal targeting pathway, and overexpressing the limiting enzyme EfmvaE significantly improved α-bisabolene production to 212 mg/L with the peroxisomal pathway. The engineered strain LH122 with the optimized peroxisomal pathway produced 1.1 g/ L α-bisabolene under fed-batch fermentation in shake flasks, achieving a 69% increase over that of the cytosolic pathway. This study provides a viable approach for overproducing isoprenoid from sole methanol in engineered yeast cell factories and shows that proteomic analysis can help optimize the organelle compartmentalized pathways to enhance chemical production.

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Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii

Cited by 12 publications

References 42 publications

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

Metabolic Engineering for Efficient Synthesis of Patchoulol in Saccharomyces cerevisiae

Engineering Yeast Peroxisomes for α-Bisabolene Production from Sole Methanol with the Aid of Proteomic Analysis

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