Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

Sun, Xiaowen; Liu, Hui; Wang, Peng; Wang, Li; Ni, Wenfeng; Yang, Qiang; Wang, Han; Tang, Hengfang; Zhao, Genhai; Zheng, Zhiming

doi:10.1186/s12934-019-1215-9

Cited by 13 publications

(12 citation statements)

References 51 publications

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“…Currently, a panel of natural product biosynthetic pathways have been reconstituted in K. phaffii, including but not limited to (+)-valencene, (+)-nootkatone, − lycopene, β-carotene, astaxanthin, , betaxanthin, menaquinone-4, lovastatin, and dammarenediol-II . Nevertheless, there has been no report on the biosynthesis of α-santalene in K.…”

Section: Introductionmentioning

confidence: 99%

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

Zuo

Xiao

Gao

et al. 2022

J. Agric. Food Chem.

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Santalene, a major component of the sandalwood essential oil, is a typical representative of sesquiterpenes and has important applications in medicine, food, flavors, and other fields. Due to the limited supply of natural sandalwood resources, there is a growing interest in engineering microbial cell factories for the mass production of santalene. In the present study, Komagataella phaffii (also known as Pichia pastoris) was established as a cell factory for high-level production of α-santalene for the first time. The metabolic fluxes were rewired toward α-santalene biosynthesis through the optimization of promoters to drive the expression of the α-santalene synthase (SAS) gene, overexpression of the key mevalonate pathway genes (i.e., tHMG1, IDI1, and ERG20), and multicopy integration of the SAS expression cassette. In combination with medium optimization and bioprocess engineering, the optimal strain (STE-9) was able to produce α-santalene with a titer as high as 829.8 ± 70.6 mg/L, 4.4 ± 0.3 g/L, and 21.5 ± 1.6 g/L in a shake flask, batch fermenter, and fed-batch fermenter, respectively. These represented the highest production of α-santalene ever reported, highlighting the advantages of K. phaffii cell factories for the production of terpenoids and other natural products.

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Section: Introductionmentioning

confidence: 99%

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

Zuo

Xiao

Gao

et al. 2022

J. Agric. Food Chem.

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“…Many synthetic biology tools have been developed to precisely control the expression of heterologous genes and the assembly and integration of multi-gene pathways in P. pastoris [ 75 ]. However, the development of P. pastoris cell factories for natural products is still limited to a few examples [ 82 ], particularly when compared with that of S. cerevisiae , indicating a need to develop novel synthetic biology tools. For example, the most commonly used promoters in P. pastoris are still pAOX1 and pGAP [ 51 ], while the construction of efficient cell factories generally requires to precisely control the expression levels of biosynthetic pathway genes.…”

Section: Discussionmentioning

confidence: 99%

“…In combination with the optimization of the fermentation conditions (i.e. pH and temperature) resulted in the maximum yield of MK-4 up to 0.24 mg/g DCW [ 82 ].…”

Section: Engineering Of P Pastoris To Produce Natumentioning

confidence: 99%

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Gao

Jiang

Lian

2021

Synthetic and Systems Biotechnology

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The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii ) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris , including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.

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“…Recently, most researchers have focused on using metabolic engineering or synthetic biology techniques to achieve a high titre, yield, and productivity (TYP) of MK-7 engineered strains. Sun et al [ 11 ] constructed a novel synthetic menaquinone-4 (MK-4) pathway in Pichia pastoris via heterologous expression of Homo sapiens UbiA prenyltransferase containing 1 (HsUBIAD1). The production of MK-4 was increased to 0.24 mg/g dry cell weight by improving geranylgeranyl diphosphate (GGPP) supply when using menadione (VK3) as the isopentenyl acceptor.…”

Section: Introductionmentioning

confidence: 99%

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

et al. 2022

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Background Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. Results We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. Conclusions In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

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Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

Cited by 13 publications

References 51 publications

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

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