Energy security and environmental pollution are two urgent and critical issues facing the world today, significantly impacting the long-term sustainable development of human society and the economy. This has led to the development of environmentally friendly energy sources as alternatives to petrochemical resources[1]. With the continuous demand for petrochemical fuels driven by economic development, it is estimated that the reserves on Earth will be depleted in approximately the next 40-50 years[2]. The use of petrochemical fuels results in environmental issues such as greenhouse gas emissions leading to the greenhouse effect and atmospheric pollution[3, 4]. Therefore, alternative energy sources need to be both sustainable and eco-friendly[5]. Scholars have researched numerous potential alternative fuels, among which bioethanol stands out as one of the most promising alternatives to petrochemical fuels due to its economic viability, renewability, and environmental friendliness. The production of bioethanol is derived from biomass, and the development of biomass energy sources globally is a crucial component of the new energy landscape[6]. Utilizing lignocellulosic biomass as raw material to produce second-generation fuel ethanol is becoming a mainstream direction for future development.

As a high-quality, clean liquid fuel, fuel ethanol has experienced rapid development internationally, particularly in countries such as the United States and Brazil. Over 50 countries and regions have implemented policies to promote the development of biofuels. The fuel ethanol industry has become an indispensable and essential part of the global energy landscape[7, 8].

Moreover, the primary raw material used in second-generation fuel ethanol production, lignocellulosic biomass, is Earth's most abundant renewable resource. Essentially, lignocellulosic biomass is a composite material composed of three major oxygen-containing macromolecular components: cellulose, hemicellulose, and lignin. Typically, these three components account for 29-47%, 25-35%, and 16-31% of the raw material composition [9, 10]. This material is highly valuable, and utilizing this renewable resource to produce high-value compounds is a strategic objective for many countries worldwide. It holds significant development potential and foresight. Conversion of lignocellulosic biomass to ethanol requires raw material pretreatment to separate xylan and lignin from crystalline cellulose. After pretreatment, crystalline cellulose remains solid, and through enzymatic hydrolysis, it is broken down into glucose, while hemicellulose is converted into xylan. Subsequently, microbial cell factories ferment glucose and xylan into ethanol. S. cerevisiae is an ideal strain for second-generation biofuel ethanol production due to its efficient metabolism of glucose to ethanol and strong tolerance to toxic compounds generated during raw material pretreatment. However, S. cerevisiae cannot metabolize xylan, the second most abundant sugar in lignocellulosic biomass[11, 12]. Therefore, developing engineered S. cerevisiae strains capable of metabolizing xylan is crucial for enhancing the economic viability of second-generation biofuel ethanol processes.

Therefore, our goal is to genetically modify the wild-type brewer's yeast to equip the strain to utilize xylan. Through rational engineering, we aim to provide theoretical support for research on fuel ethanol production.

cerevisiae utilizes xylose by first importing it into exogenous oxidation-reduction or isomerization pathways, converting xylose into xylulose that yeast can metabolize. The oxidation-reduction pathway relies on the actions of xylose reductase and xylitol dehydrogenase[11, 13] (Figure 1). However, their different cofactor preferences lead to significant accumulation of the intermediate xylitol, severely impacting downstream reactions. The isomerization pathway, catalyzed by xylose isomerase (XI), directly converts xylose into xylulose in a single step (Figure 1). This pathway is preferred for constructing xylose-fermenting yeast due to its simplicity and absence of cofactor imbalance issues. Additionally, overexpression of the endogenous xylulokinase gene XKS1 and genes in the pentose phosphate pathway (PPP) can further enhance the metabolic flux of xylose into the sugar fermentation pathway. Subsequent xylose medium domestication results in yeast strains with significantly improved xylose utilization phenotypes[14]. This simple molecular manipulation combined with domestication has become the paradigm for rapidly developing xylose-metabolizing yeast. Through reverse metabolic engineering strategies, comparative genomic analyses of strains before and after domestication can identify candidate differential molecular mutations. Subsequent genetic experiments involving knockout and complementation are used to validate whether these candidate mutations promote xylose utilization. Proposed hypotheses on the beneficial molecular mutation mechanisms are then tested, leading to iterative strain design and construction. Rational construction of xylose-utilizing yeast is achievable through this approach.

Therefore, based on the principles of synthetic biology, this study utilizes molecular biology techniques to perturb genes related to iron ion metabolism within S. cerevisiae, testing their impact on the yeast's ability to utilize xylose. By identifying molecular targets that enhance S. cerevisiae metabolism of xylose, this research aims to contribute to the bioethanol industry using lignocellulosic biomass as a fuel source.

Figure 1. The schematic diagram of xylose metabolism

This study, based on the concept of synthetic biology, aims to construct a strain of brewer's yeast capable of simultaneously metabolizing xylose using molecular biology methods. By endowing the strain with the ability to utilize multiple substrates for ethanol production, effective targets for enhancing xylose utilization in the strain will be screened and identified. The goal is to provide a research foundation for the development of fuel ethanol production.

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