Recommended literature of synthetic biology: peroxisome metabolic coupling is combined into fatty alcohol
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- Time of issue:2023-04-17 13:36
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(Summary description)Summary The efficient utilization and biotransformation of one-carbon compounds are the current research hotspots in the field of biomanufacturing. Among them, methanol can be prepared from CO2 in large quantities, so it is considered to be an environmentally friendly biomanufacturing raw material with potential. Due to the cytotoxicity of methanol and formaldehyde
Recommended literature of synthetic biology: peroxisome metabolic coupling is combined into fatty alcohol
(Summary description)Summary
The efficient utilization and biotransformation of one-carbon compounds are the current research hotspots in the field of biomanufacturing. Among them, methanol can be prepared from CO2 in large quantities, so it is considered to be an environmentally friendly biomanufacturing raw material with potential. Due to the cytotoxicity of methanol and formaldehyde
- Categories:Industry News
- Author:
- Origin:
- Time of issue:2023-04-17 13:36
- Views:
Information
Summary
The efficient utilization and biotransformation of one-carbon compounds are the current research hotspots in the field of biomanufacturing. Among them, methanol can be prepared from CO2 in large quantities, so it is considered to be an environmentally friendly biomanufacturing raw material with potential. Due to the cytotoxicity of methanol and formaldehyde (the oxidation intermediate of methanol), it is challenging to build a microbial cell factory that can bioconvert methanol into a target product with high efficiency. Recently, the team of Zhou Yongjin, a researcher at the Dalian Institute of Chemical Sciences, Chinese Academy of Sciences,used the peroxisome compartmentalization strategy in Ogataea polymorpha to couple methanol metabolism and fatty alcohol synthesis pathway to achieve efficient bioconversion of methanol to fatty alcohol. This paper provides a feasible engineering strategy for the study of improved methanol biotransformation.
Research content
Fatty alcohols are widely used in detergents, detergents, and emollients in the field of toiletries, and the global fatty alcohols market is expanding year by year, and is expected to reach 2025 billion US dollars in 70. A variety of microorganisms have been used to build microbial cell factories that bioconvert from one-carbon compounds to fatty alcohols (Table 1).
1. Construction of fatty alcohol synthesis pathway
The synthesis of fatty alcohols can be mainly reduced by fatty acyl-CoA or free fatty acids in three pathways (Fig.1A). The authors first compared the efficiency of these three pathways in synthesizing fatty alcohols in Hanson's yeast. The TaFAR1 pathway exhibits a 25-fold higher amount of fatty alcohol synthesis than other pathways (Fig.1B), so this synthetic pathway is used for follow-up studies in this article
In addition to TaFAR1, this pathway requires the involvement of alcohol dehydrogenase/aldehyde reductase (ADH/ALR) to convert fatty aldehydes to fatty alcohols. In order to improve the conversion efficiency of fatty aldehyde to fatty alcohol, the authors expressed Saccharomyces cerevisiae-derived ScADH5, which increased the fatty alcohol yield of Hanson's yeast by 34% ±10%, which also showed that the reduction of fatty aldehyde was a bottleneck reaction in the fatty alcohol synthesis pathway. After overexpression/knockout testing of endogenous ADH in Hanson's yeast, the authors found that knockout of ADH6 and ADH7 genes significantly increased fatty alcohol production, with ADH6-3 achieving the highest fatty alcohol production, indicating that these endogenous alcohol dehydrogenases hinder fatty alcohol synthesis. The combination of overexpression of ScADH5 and knockout of ADH6-3 (ZX-F15) resulted in a titer of fatty alcohols of (158±8) mg/L, which was 18%±106% higher than ZX-F9 [(49±7) mg/L] (Fig.1C). From the perspective of genetic stability, the authors integrated TaFAR1 and ScADH5 into the genome of Hanson's yeast (ZX-F33), which exhibited lower fatty alcohol yield and biomass in methanol medium compared with glucose medium. This phenomenon indicates that when methanol is used as a carbon source for fatty alcohol synthesis, the fatty alcohol synthesis pathway in the cytosol is not suitable (Fig.1D). Therefore, when constructing methanol biotransformation cell factories, it is not critical to coordinate methanol metabolism and product synthesis.
1. Construction of peroxisome compartmentalized methanol-to-fatty alcohol synthesis pathway
Hanson's yeast is a methyl-trophic yeast whose peroxisome is the main site of methanol assimilation and fatty acid β-oxidation. Peroxisome compartmentalization constructs the synthesis pathway from methanol to fatty alcohol, which can effectively avoid the interference of its own cytosolic fatty acid homeostasis, and can also couple methanol metabolism with fatty alcohol synthesis. Whether it is a constitutive promoter (pGAP) or a methanol-induced promoter (pTAL1-2), the fatty alcohol synthesis pathway of peroxisomes has a fatty alcohol yield 3.9 times higher than the cytosolic pathway (Fig.2B). Consistent with the results of the cytosolic synthesis pathway, the overexpression of ScADH1 by peroxisomes on the strain integrating TaFAR41 (ZX-F5) yielded ZX-F39, and the fatty alcohol yield was also increased by 16% ±7%. However, when genome integration of 2 copies of TaFAR1 and ScADH5, the fatty alcohol yield of ZX-F40 and OD value bacteria decreased significantly (Fig.2C), indicating that enhancing the fatty alcohol synthesis pathway may disrupt methanol metabolism and homeostasis of fatty acyl-CoA, affecting normal cell growth.
To further increase the supply of the precursor fatty acyl-CoA, HFD39 (encoding fatty aldehyde dehydrogenase), ARE (encoding acetyl-CoA: gallbladder) were knocked out sequentially on the ZX-F1 strain
Sterol acyltransferase), LPL1 and IZH3 (related to phospholipid homeostasis), overexpression of PXA1,2 (encoding peroxidase lipyl-CoA transporter), resulting in a gradual increase in fatty alcohol production. The fatty alcohol yield of ZX-F51PXA reached (131±1) mg/L (Fig.2D). To test the stability of the strains, ZX-F51 and ZX-F51PXA were fermented in 2 0 g/L methanol medium, which increased yields by 10%, ±64%, 11±%, and 79%, respectively, compared to the yield in 15 g/L methanol medium (Fig.2E).
