Difference between revisions of "Team:ShanghaiFLS China/Description"

In the 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 subply. (Dürre & Eikmanns, 2015) Moreover, compared to conventional carbon substrates in the biotech industry (e.g. glucose), single-carbon compounds areespecially preferable for their abundance and their ubiquitous presence in industry exhausts, potentially enabling the conversion ofwaste to not only fuel, but also a myriad of possible commercial and medicinal compounds.

Methanol specifically, is not only a major byproduct of China’s magacoal industry (coal-produced methanol accounts for 77% of the total methanol production in China, and China’s methanol production capacitiesaccounts for 58% of the global production)(中国产业信息网, 2018; 隆众聚焦, 2018), but can also be readily converted from synthesis gases (syngas), most commonly acquired from industry exhausts.

Structural formula of methanol, via Wikipedia.org

Pichia pastoris, a 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 (P_AOX1) is characterized in detail for the first time.

Left: The methanol metabolic pathways of P. pastoris. Alcohol oxidase 1 is one of the two peroxisomes that converts methanol into formaldehyde, which is then further metabolized. Adapted from Vogl et al., 2016. Right: P_AOX1 regulation. Adapted from X. Wang et al., 2016. P_AOX1 is activated by a cascade of transcription factors Mxr1, Prm1, and Mit1: Mxr1 is essential for P_AOX1 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 P_AOX1, while Prm1 and Mit1 strongly activate P_AOX1, upregulating the expression of alcohol oxidase 1. Besides activating P_AOX1 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(J. 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.

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