Construction of an orthogonal transport system for Saccharomyces cerevisiae peroxisome to efficiently produce sesquiterpenes.

Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.

Metabolic Engineering of Saccharomyces cerevisiae for Heterologous Carnosic Acid Production.

Carnosic acid (CA), a phenolic tricyclic diterpene, has many biological effects, including anti-inflammatory, anticancer, antiobesity, and antidiabetic activities. In this study, an efficient biosynthetic pathway was constructed to produce CA in Saccharomyces cerevisiae. First, the CA precursor miltiradiene was synthesized, after which the CA production strain was constructed by integrating the genes encoding cytochrome P450 enzymes (P450s) and cytochrome P450 reductase (CPR) SmCPR. The CA titer was further increased by the coexpression of CYP76AH1 and SmCPR ∼t28SpCytb5 fusion proteins and the overexpression of different catalases to detoxify the hydrogen peroxide (H2O2). Finally, engineering of the endoplasmic reticulum and cofactor supply increased the CA titer to 24.65 mg/L in shake flasks and 75.18 mg/L in 5 L fed-batch fermentation. This study demonstrates that the ability of engineered yeast cells to synthesize CA can be improved through metabolic engineering and synthetic biology strategies, providing a theoretical basis for microbial synthesis of other diterpenoids.

Research progress in bioremediation of petroleum pollution.

With the enhancement of environmental protection awareness, research on the bioremediation of petroleum hydrocarbon environmental pollution has intensified. Bioremediation has received more attention due to its high efficiency, environmentally friendly by-products, and low cost compared with the commonly used physical and chemical restoration methods. In recent years, bacterium engineered by systems biology strategies have achieved biodegrading of many types of petroleum pollutants. Those successful cases show that systems biology has great potential in strengthening petroleum pollutant degradation bacterium and accelerating bioremediation. Systems biology represented by metabolic engineering, enzyme engineering, omics technology, etc., developed rapidly in the twentieth century. Optimizing the metabolic network of petroleum hydrocarbon degrading bacterium could achieve more concise and precise bioremediation by metabolic engineering strategies; biocatalysts with more stable and excellent catalytic activity could accelerate the process of biodegradation by enzyme engineering; omics technology not only could provide more optional components for constructions of engineered bacterium, but also could obtain the structure and composition of the microbial community in polluted environments. Comprehensive microbial community information lays a certain theoretical foundation for the construction of artificial mixed microbial communities for bioremediation of petroleum pollution. This article reviews the application of systems biology in the enforce of petroleum hydrocarbon degradation bacteria and the construction of a hybrid-microbial degradation system. Then the challenges encountered in the process and the application prospects of bioremediation are discussed. Finally, we provide certain guidance for the bioremediation of petroleum hydrocarbon-polluted environment.