Cholesterol, an essential lipid for mammalian cells, can be used as a carbon source by bacteria and as a precursor for steroid hormones in animal cells. The cholesterol C17 side-chain-cleavage pathways are the committed and rate-limiting steps in the biosynthesis of steroid drugs. Three cholesterol C17 side-chain degradation pathways have been identified in nature: the β-oxidation pathways in actinobacteria, the oxygen-independent degradation pathway in Sterolibacterium denitrificans, and the cholesterol side-chain degradation pathway in mammals. An in-depth understanding of the structures and molecular mechanisms of enzymes in these degradation processes will facilitate the creation of enzyme mutants with better catalytic capabilities. The introduction of the engineered enzymes into the microbial cell factories may contribute to the industrial production of steroid drugs.
With staggering progress on genetic manipulation strategies, Saccharomyces cerevisiae is becoming an ideal cell factory for the de novo biosynthesis of lipid compounds. However, due to their hydrophobicity, lipids tend to be accumulated within intracellular spaces and cause a high burden on cell activity and induce product inhibition effect, which ultimately restricted the lipids biomanufacturing for industrial application. Herein, an oleic acid stress (OAS) model was applied for the long-time domestication of BY4741 cells, and a subclone of A-22 was obtained through a series of acclimation (0.1% glucose and 0.2% oleic acid), showing increased accumulation of both biomass and intracellular lipid droplets compared to WT. Comparative transcriptome analysis indicated that compared to fatty acid metabolism, most transcripts enriched in the pathways of glucose catabolism (glycolysis and citrate cycle) and lipid synthesis (phospholipid and sterol) were down-regulated under OAS. While interestingly, most the above transcripts tended to be ‘restored’ in adapted strain A-22. In addition, for physical adaptation, significant increase of phosphatidylcholines was identified by lipidomic analysis, which probably caused the subsequent subcellular expansion of peroxisomes and lipid droplets as observed in the adapted strain, since phosphatidylcholines are the major constituent of their membranes. The present study systematically investigated both the phenotype change and molecular mechanism on adaptation of S. cerevisiae towards oily environment. Detailed information on functional transcripts may provide novel rational modification targets to reinforce the hydrophobic lipids biosynthesis within S. cerevisiae engineered cell factory.
With the increasing application of steroid drugs as therapeutics, the demand for steroid drugs is increasing. In recent years, biological synthesis has become the standard approach to produce steroid intermediates, while this method still faces some problems such as unclear metabolic pathway and low yield. Mycobacterium sp. LY-1 can convert phytosterols into 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) which is a key intermediate for the synthesis of steroid drugs with long effective time and significant pharmacological activity. In this work, the whole-genome sequence of the Mycobacterium sp. LY-1 was analyzed, and the side-chain degradation pathway of phytosterols in Mycobacterium sp. LY-1 was proposed. Meanwhile, the related key enzymes of phytosterol metabolism were identified through qRT-PCR. Through overexpressing the key enzymes including KshA2, KshB, and HsdB, the yield of 9α-OH-AD increased by 12.7% compared to that of the control. Furthermore, by optimizing the medium and culture conditions, the yield of 9α-OH-AD reached 50.4%. The maximum yield was 30.7% higher than that of the original strain. The results are of significance for the industrial production of 9α-OH-AD using metabolic engineering methods.