Metabolic engineering of Saccharomyces cerevisiae for chelerythrine biosynthesis.

BACKGROUND: Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming. RESULTS: The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain. CONCLUSIONS: This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.

Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.

BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

[Bacteria in super capacitor electrode materials: a review].

As a major factor in determining performances of the electrochemical super capacitor, electrode materials have received wide attentions recently. Bacteria, with their advantages of low cost, abundance, environmental protection and easy to get from nature, have become the promising biomaterials. The composite electrode materials based on bacteria or their related products, have the advantages of large specific surface area, good cycle stability and high capacitance, and become the research focus in the field of super capacitor electrode materials. Herein, the characteristics and related technology of different bacteria on the preparation of electrode materials for super capacitor were summarized. This review systematically elaborates the latest research progress of bacterial composite electrode materials. Moreover, the prospect of super capacitors has been discussed.