Xiao-Man Sun

Fine‐tuning the expression level of multigene pathways remains a major challenge in engineering eukaryotic microbial cell factories. Modifications to individual pathway genes and CRISPR‐based approaches often require repeated genome reconstruction, large regulatory components, or complex guide‐array assembly. Here, we developed a lightweight, post‐transcriptional RNAi‐based strategy that enables fine‐tuning gene expression without iterative genome editing. Size‐selected small‐RNA sequencing and functional testing were used to identify endogenous Pol III promoters spanning graded transcriptional strengths in Schizochytrium sp. Combining promoter strength with multiple short hairpin RNA (shRNA) target positions expanded the accessible repression range and enabled high‐resolution gene‐dosage tuning. The RNAi‐based combined regulation strategy was applied to the three‐gene carotenoid pathway of Schizochytrium sp. and the nine‐gene docosahexaenoic acid (DHA) pathway of Yarrowia lipolytica , significantly enhancing the product production in both hosts. Integration of RNAi screening and dosage optimization further identified and tuned bypass pathways, increasing the ratio of DHA to docosapentaenoic acid (DPA) by 4.50‐fold versus wild type. These gains were maintained during 5‐L fermentation and serial passaging. This work establishes a compact and scalable regulatory framework for engineering complex biosynthetic pathways in eukaryotic microbial hosts.

Despite the widespread industrial applications of nanozymes, their usage is often limited by mechanistic factors. They lack reaction control with catalyst fouling, which limits catalytic turnover and makes nanozyme recovery an energy-intensive process at large scale. These mechanistic challenges have been addressed by the evolution of photoresponsiveness into nanozymes called as photonanozymes (PNZs). This research is focused to critically assess the architecure of photonanozymes leading to photoresponsivness. A three domain framework deconstructing photoresponsive mechanism with research road map to guided subsequent investigation constitutes the distinctive contribution of this review. The photoresponsive architechture elucidates the mechanism of light gated catalysis governed by wavelength selectivity and spatiotemporal precison. Thereby, expanding the enzyme mimicking catalysis and its applciations in environmental remediation, theragnostics, cancer therapy, biosensing and biofuel synthesis. Rather than merely describing the operating principles of photonanozymes, rational foundation is build up which determines future design. Challenges impeding photocatalytic mechanisms are critically examined including limited wavelength selectivity, insufficient opertional stability and difficulty of achieving reproducible and scalable synthesis. Presented review strategizes a forward-looking roadmap centered around robust PNZ design, standardized performance, and translation of light-programmable catalysis from laboratory proof-of-concept to practical technology. Through a rigorous and comprehensive examination of inherent complexities, it frames PNZs as a transformative step towards programmable, self-healing nanomaterials with controlled implications, serving as an invaluable resource for those seeking orientation within photocatalysis. Furthermore, it reaches beyond its immediate boundaries, presenting new ideas, evidence-based conclusions, and prospective directions intended to influence the course of future investigations.

Docosahexaenoic acid (DHA) is essential for human health, and sustainable production of DHA lipids by Schizochytrium sp. is industrially important. Unlike conventional bottom-up engineering of individual pathway enzymes, this study applied a top-down strategy using heterologous transcription factors. Screening identified soybean GmDof4 as the most potent regulator. Overexpression significantly boosted total lipid yield by 31.7% in a 5-L bioreactor and improved glucose conversion efficiency by 22.4%. Although the DHA proportion decreased slightly, absolute DHA lipid titer and yield rose by 20.8% and 14.4%, respectively. Mechanistically, GmDof4 primarily upregulated acetyl-CoA carboxylase (ACCase) while coordinately modulating fatty acid synthesis and triacylglycerol assembly. Chemical inhibition of ACCase abolished this enhancement, confirming its centrality. These findings validate transcription factor engineering as a powerful strategy for developing high-performance microbial DHA platforms.

Docosahexaenoic acid (DHA), an important ω3 polyunsaturated fatty acid (PUFA), is widely applied in health products and infant formulas due to its significant physiological functions for human health. The traditional sources of deep-sea fish and shrimp extraction have limited industrialization and sustainable synthesis. Yarrowia lipolytica is an excellent host for PUFA synthesis. However, there are limited reports on DHA production in Y. lipolytica. In this study, Y. lipolytica was engineered to successfully produce DHA through systematic metabolic engineering, including the screening of high-efficiency elongase/desaturase, increasing the copy number of key genes, and promoting the storage of DHA by substituting acyltransferases with DHA-CoA preference. Finally, the DHA titer of the engineered strain reached up to 9.06 g/L, with a content of 36.8% in a 5 L bioreactor, representing the highest yield reported to date in yeast. This study broadened the methods for the green and sustainable synthesis of DHA.