Application of adaptive laboratory evolution to improve the tolerance of <i>Rhodotorula</i> strain to methanol in crude glycerol and development of an effective method for cell lysis

Wei, Shiyu; Wang, Hongyang; Fan, Meixi; Cai, Xinrui; Hu, Junpeng; Zhang, Ruixin; Song, Baocai; Li, Jing

doi:10.1002/biot.202300483

Cited by 5 publications

(3 citation statements)

References 39 publications

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“…pastoris. Though the α-bisabolene production from methanol was lower than that from glucose, ,− further engineering central metabolism , and enhancing the methanol tolerance , should be helpful for improving the cellular performance for chemical production.…”

Section: Discussionmentioning

confidence: 99%

“…In summary, we comprehensively optimized and compared the MVA pathway in the peroxisome and cytosol to produce sesquiterpene α-bisabolene from the sole methanol in P. pastoris . Though the α-bisabolene production from methanol was lower than that from glucose, 1 , 3 − 5 further engineering central metabolism 33 , 35 and enhancing the methanol tolerance 44 , 45 should be helpful for improving the cellular performance for chemical production.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

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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“…[19] Wei et al improved the tolerance of Rhodotorula toruloides to methanol in crude glycerol by laboratory evolution, thus utilizing crude glycerol as a low-cost carbon source for lipid production. [20] However, there have been no reported findings on the utilization of glycerol as a sole carbon source for terpenoid production in S. cerevisiae.…”

mentioning

confidence: 99%

Engineering and transcriptome study of Saccharomyces cerevisiae to produce ginsenoside compound K by glycerol

Zhang,

Tian,

Zhang

et al. 2024

Biotechnology Journal

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Synthetic biology‐based engineering of Saccharomyces cerevisiae to produce terpenoid natural products is an effective strategy for their industrial application. Previously, we observed that glycerol addition was beneficial for ginsenoside compound K (CK) production in a S. cerevisiae when it was fermented using the YPD medium. Here, we reconstructed the CK synthesis and glycerol catabolic pathway in a high‐yield protopanaxadiol (PPD) S. cerevisiae strain. Remarkably, our engineered strain exhibited the ability to utilize glycerol as the sole carbon source, resulting in a significantly enhanced production of 433.1 ± 8.3 mg L−1 of CK, which was 2.4 times higher compared to that obtained in glucose medium. Transcriptomic analysis revealed that the transcript levels of several key genes involved in the mevalonate (MVA) pathway and the uridine diphosphate glucose (UDPG) synthesis pathway were up‐regulated in response to glycerol. The addition of glycerol enhanced CK titers by augmenting the flux of the terpene synthesis pathway and facilitating the production of glycosyl donors. These results suggest that glycerol is a promising carbon source in S. cerevisiae, especially for the production of triterpenoid saponins.

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Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L‐cysteine

Yang,

Zhang,

et al. 2024

Biotechnology Journal

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L‐Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L‐cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L‐cysteine tolerance in Escherichia coli. ARTP‐treated populations obtained substantial enhancement in L‐cysteine tolerance by ALE. Whole‐genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L‐cysteine resistance. The isolated tolerant strain, 60AP03/pTrc‐cysEf, achieved a 2.2‐fold increase in L‐cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L‐cysteine production. The production evaluations, supplemented with L‐serine, further demonstrated the stability and superiority of tolerant strains in L‐cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L‐cysteine, providing valuable insights into improving L‐cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.

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Application of adaptive laboratory evolution to improve the tolerance of Rhodotorula strain to methanol in crude glycerol and development of an effective method for cell lysis

Cited by 5 publications

References 39 publications

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

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

Engineering and transcriptome study of Saccharomyces cerevisiae to produce ginsenoside compound K by glycerol

Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L‐cysteine

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