Team:ShanghaiFLS China/Description
Project Inspiration
In recent years, single-carbon compounds (e.g., methanol, methane, carbon dioxide, carbon monoxide, etc.) have gained much attention as an alternative to fossil fuels for sustainable fuel supply. (Dürre & Eikmanns, 2015) Compared to conventional carbon substrates in the biotech industry, single-carbon compounds are especially preferable for their abundance and their ubiquitous presence in industry exhausts, which could have been a pollutant or a useless byproduct otherwise.
Structural formula of methanol, adapted from Wikipedia.org
Coal-produced methanol accounts for 77% of the total methanol production in China, and China’s methanol production capacities account for 58% of the global production (中国产业信息网, 2018; 隆众聚焦, 2018). Moreoevr, methanol can be readily converted from synthesis gases, most commonly acquired from industry exhausts.
Goals and Design
Pichia pastoris GS115, a strain of methylotrophic yeast, is capable of utilizing methanol as its substrate and is awidely used expression system of heterogeneous proteins.(Cereghino& Cregg, 2000; Gasser et al., 2013) This is enabled by the highly specific regulation of the AOX1 gene that codes for alcohol oxidase 1, the key enzyme in the methanol metabolic pathway. Recently, the in trans regulation of the AOX1 promoter (PAOX1) is characterized in detail for the first time.
Left: The methanol metabolic pathways of P. pastoris. Alcohol oxidase 1 is the key enzyme that converts methanol into formaldehyde, which is then further metabolized. Adapted from Vogl et al., 2016. Right: PAOX1 regulation. Adapted from Wang et al., 2016. PAOX1 is activated by a cascade of transcription factors Mxr1, Prm1, and Mit1: Mxr1 is essential for PAOX1 de-repression and is inhibited by glucose. When methanol is present as the only carbon source, however, Mxr1 is derepressed, and Prm1 expression is induced by methanol. Prm1 expression is further amplified by its self-activation, while Mit1 expression is also upregulated by Prm1 activation. Taken together, Mxr1 derepresses PAOX1, while Prm1 and Mit1 strongly activate PAOX1, upregulating the expression of alcohol oxidase 1. Besides activating PAOX1 though, Mit1 also represses the expression of Prm1, down regulating the cascade overall.
In earlier research, the P. pastoris GS115 strain had been modified to produce medicinal products such as the insulin precursor(Wang et al., 2017), lovastatin and monacolin J (a precursor of simvastatin) (both lovastatin and simvastatin are widely prescribed antihypertensive drugs)(Liu et al., 2018).
Lovastatin and simvastatin synthesis pathways in engineered P. pastoris GS115, adapted from Liu et al., 2018. By co-culturing two engineered P. pastoris GS115 strains that share the pathwayin methanol media, Liu et al. was able to achieve a 250.8 mg/L yield of lovastatin and a 593.9 mg/L yield of monacolin J. This is considered much more preferable than the conventional fermentation by native fungi such as A. terrus, which requires long incubation time, and produces multiple byproducts.
The metabolization of methanol by P. pastoris GS115, however, has its own limitations. It consumes much oxygen and releases much heat, which has posed higher requirements on the fermentation equipment. We therefore attempted to create modified strains of P. pastoris GS115 that are more efficient at metabolizing methanol. i.e. strains which are capable of producing the same amount of product while consuming less methanol, hence consuming less oxygen and releasing less heat.
In order to further upregulate PAOX1, we aim to recombine the homologous promoters and transcription factors as our composite parts and integrate them into the wild type P. pastoris GS115 strain, which shall not only additionally activate PAOX1, but also positively regulate the expression levels the homogeneous Mit1 and Prm1. We will use yEGFP3 (abbr. GFP), a yeast optimized EGFP variant as our reporter gene of PAOX1 activity.
Significance
Compared to optimizing the industrial fermentor, our approach via synthetic biology has its unique advantage in that it doesn't require any reinstallation of hardware in the production plants, significantly reducing the cost for process optimization: the manufacturers only need to change the strain of yeast they are using, the rest mostly stays the same. This is especially preferable for large-scale fermentor productions given that in such plants the hardware constitutes a significant portion of assets. Moreover, as we are optimizing the regulatory pathways upstream from the product synthesis pathways, technically our approach can be applied to all industrial applications of methanol fermentation via P. pastoris GS115, ranging from biofuel production to medicine synthesis. In short, if successful, our methods via synthetic biology can be a very elegant solution to the excessive heat generation problem of P. pastoris GS115 fermentation with virtually unlimited potentials.
References
Cereghino, J. L., & Cregg, J. M. (2000, January). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews. https://doi.org/10.1016/S0168-6445(99)00029-7
Dürre, P., & Eikmanns, B. J. (2015, December 1). C1-carbon sources for chemical and fuel production by microbial gas fermentation. Current Opinion in Biotechnology. Elsevier Ltd. https://doi.org/10.1016/j.copbio.2015.03.008
Gasser, B., Prielhofer, R., Marx, H., Maurer, M., Nocon, J., Steiger, M., … Mattanovich, D. (2013). Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiology, 8(2), 191–208. https://doi.org/10.2217/fmb.12.133
Liu, Y., Tu, X., Xu, Q., Bai, C., Kong, C., Liu, Q., … Cai, M. (2018). Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol. Metabolic Engineering, 45(June 2017), 189–199. https://doi.org/10.1016/j.ymben.2017.12.009
Vogl, T., Sturmberger, L., Kickenweiz, T., Wasmayer, R., Schmid, C., Hatzl, A. M., … Glieder, A. (2016). A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris. ACS Synthetic Biology, 5(2), 172–186. https://doi.org/10.1021/acssynbio.5b00199
Wang, J., Wang, X., Shi, L., Qi, F., Zhang, P., & Zhang, Y. (2017). Methanol-Independent Protein Expression by AOX1 Promoter with trans -Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris. Nature Publishing Group, (December 2016), 1–12. https://doi.org/10.1038/srep41850
Wang, X., Wang, Q., Wang, J., Zhou, M., Shi, L., Zhou, X., … Shen, W. (2016). Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 ( AOX1 ) Promoter in Pichia pastoris. Journal of Biological Chemistry, 291(12), 6245–6261. https://doi.org/10.1074/jbc.m115.692053
中国产业信息网. (2018). 2017年中国甲醇行业发展现状及价格走势分析. Retrieved October 17, 2019, from https://www.chyxx.com/industry/201805/640922.html
隆众聚焦. (2018). 2016年全国甲醇原料生产分布及2017年新增产能占比分析. Retrieved October 17, 2019, from https://m.baidu.com/ala/c/www.360doc.cn/mip/737049813.html