Description

Compared to chemical manufacturing, bio-manufacturing is environmentally friendly and efficient, making it a promising and future approach fitting the theory of sustainable development and production. The selection of suitable cell factories for production is crucial in bio-manufacturing, and researchers should consider factors such as ease of cell cultivation, degree of chassis standardization, and capacity of potential production. Among commonly used cell factories, such as Escherichia coli, Bacillus subtilis, microalgae, and CHO cells, Saccharomyces cerevisiae cell factories possess numerous advantages, including extensively characterized genomic information and high degree of chassis standardization. Recently, many studies have demonstrated the production capacity of S. cerevisiae cell factories by producing biofuels¹, aromatic compounds² and so on, highlighting their significant development potential.

However, S. cerevisiae cell factories still face numerous challenges, with insufficient stress tolerance being a major concern that directly affects cell viability and product yield. These stresses arise from both biotic factors, such as microbial contamination and viral infections, and abiotic factors, such as pH fluctuations, unsuitable temperature and toxic compounds. During production, creating a sterile environment or adding antagonistic factors to the fermentation broth are commonly used methods to address these difficulties. However, these methods consume a amount of energy, contradicting the principles of green manufacturing and hindering sustainable development.

One specific case involves the fermentation of ethanol using straw as a resource. In this process, physical pretreatment methods such as steam explosion are often employed to break down the straw. However, this results in a rapid increase in the temperature of the system. To facilitate subsequent fermentation, the system needs to be quickly cooled down using cooling water. The production of cooling water consumes a significant amount of energy and increases production costs. Therefore, it is more commendable to enhance the stress tolerance(for this case, that is the thermo-tolerance) of S. cerevisiae cell factories through synthetic biotechnology, which caused the amount of cooling water required to be reduced, leading to a decrease in energy consumption and an increase in production profitability.

Polyamines are a group of organic compounds that contain amino groups. They are derived from secondary metabolic pathways within organisms and play important roles in various physiological activities. Short- and linear- chain polyamines like putresine and spermidine are commonly exist in most species, however, long- and branch-chain polyamines exist trace in organisms but function importantly. For example, Thermus thermophilus, an extreme thermophile, can grow from 47℃-85℃, and produces 16 different polyamines including long-chain and branched-chain polyamines. With temperature raised, the amount of long- and branch-chain polyamines are increased, which indicates that there is a strong relationship between environment temperature and the regulation of polyamines³. Additionally, Sun, K et al.⁴ demonstrated improved thermotolerance as well as resistance to acid, furan, and other compounds in Clostridium thermocellum by overexpressing an endogenous spermidine synthase and a butanol dehydrogenase from Thermoanaerobacter pseudethanolicus. This work indicated us the tolerance to stress is not a robustness mode, but a complex network, which means any tolerant mechanism can be related to different stresses.

Qin, J et al.⁵ reprogrammed the metabolism of baker’s yeast S. cerevisiae and recruited nature’s diverse reservoir of biochemical tools to enable a complete biosynthesis of multiple polyamines and polyamine analogues. This paper gave us many resources to engineer polyamine metabolic and tune relationship of quantity and effect. For our project, we paid more attention to the synthesis of long-chain polyamines. To easily understand the formation of different polyamines, we can view this process as addition aminopropyl by aminopropionyltransferase in different C position of the basic polyamine skeleton, which is putresine usually. Extend from here, we also consider to dig aminopropionyltransferases from different species by bioinformatics, especially extreme thermophiles.

Overall, this inspires us to consider polyamines and their derivatives as potential stress-tolerant parts to address the limited stress tolerance of S. cerevisiae cell factories.

Considering the important role of heat in bio-fermentation, 2023 SCU-BES-China aims to enhance the thermotolerance of S. cerevisiae cell factories by manipulating the expression of different kinds of polyamines, such as spermidine and thermospermine.

We achieved our goal from four aspects.

First, we planed simply to add different concentration of spermidine into culture media and investigate whether spermidine confers thermo-tolerance capabilities to wild type strain.

Second, we planed to use CRISPR/Cas9 to replace the endogenous OAZ1 in S. cerevisiae with the SPE1 or SPE1-AtACL5 to weak the effect of OAZ1, which encodes an antizyme of SPE1 and decrease directly the amount of putresine. And the SPE1 or SPE1-AtACL5 can confer synthesis ability of putresine and thermospermine to wild type strain. Then we will examine its thermo-tolerance capabilities.

Third, we planed to express SPE1(which is from yeast itself and can increase the accumulation of putresine, the detailed information of this gene can be found in our part page), AtACL5(which is from Arabidopsis thaliana and can increase the accumulation of thermospermine, the detailed information of this gene can be found in our part page) and both by plasmid in OAZ1Δ strain and to examine its thermo-tolerance capabilities.

Fourth, we planed to fine-tune the expression levels of SPE2(which is a S-adenosylmethionine decarboxylase, catalysing SAM into dSAM. dSAM provides the source of the propylamine group, which is the foundation of polyamine.), SPE3(In yeast, spermidine is formed by the addition of a propylamine moiety to putrescine, catalysed by an aminopropyltransferase termed spermidine synthase (SPDS, namely SPE3), with S-methyl-5’-thioadenosine (MTA) as a co-product.), and AtACL5 by plasmid in the OAZ1Δ::SPE1 strain and subsequently assessed their thermo-tolerance capabilities.