Study On The Regulation Of Osmolarity On Key Metabolic Genes Of C.glycerinogenes

Candida glycerinogenes is an excellant stress resistance industrial strain, the key enzymes, phosphofructokinase(PFK), pyruvate kinase(PYK) and glucose-6-phosphate dehydrogenase(G6PDH), from EMP and HMP pathways, as well as the key enzyme of glycerol production, cytoplasmic glycerol-3-phosphate dehydrogenase(ctGPD), highly contribute to the osmoadaption and glycerol accumulation of yeast. However, the regulation mechanism of osmolarity on these enzymes remains ambiguous. This study aims at understaning the regulation mechanism of osmolarity on the enzymes from fermentation, protein and transcription levels.Putative genes of Cgpfk1, Cgpfk2, Cgzwf and Cgpyk were cloned from genomic DNA. The S. cerevisiae pfk1? and S. cerevisiae pyk1? mutants were constructed by PCR-mediated Tecchnology. Based on these strains and the lab stored S.cerevisiae pfk2 and S. cerevisiae zwf?, the physiological functions of Cgpfk1, Cgpfk2, Cgzwf and Cgpyk were confirmed by complemtntation and enzymatic analysis. These results establised the foundation for further study.Fermentation analysis showed that low osmotic stress(20 g·L-1 NaCl) had no impact on cell growth, except for a 4 h lag phase. The glcerol titer reached 1.7 times as control(salt-free). High osmotic pressure(50 g·L-1 NaCl) postphoned cell growth by 12 h but had on inhibition on maximal biomass and led to higher glycerol titer(3 times as control). Metabolic flux analysis showed that osmolarity forced more glucose flux flowed into EMP pathway from HMP pathway and carbon flux into lactose, ethanol, pyruvate pathways and TCA cycle decreased in glycerol production phase. In conparison, high osmotic pressure trigger bigger deviation of carbon flux of above trendency.In contrast, PFK and ctGPD exhibited higher activity(up to 1.6 and 1.3 times), G6 PDH activity remained slightly lower(about 80%), PYK activity markably increased when the strain treated with NaCl. The enzyme activities changing infer that osmolarity enhanced general carbon flux but decreasd the flux into TCA cycle. Meanwhile, the significantly increased ctGPD activity forced carbon flux to glycerol pathway to against the osmotic stress. G6 PDH activity remained slower when pressured with osmolarity and cell growth hence retarded. The enzyme regulation aggravated with higher osmolarity.Transcription analysis showed results as follows: Cggpd upregulated with osmolarity to the maximum fold change of 7.7. Nevertheless, Cgpfk1, Cgpfk2, Cgzwf and Cgpyk showed unremarkable fold change. It infer that C. glycerinogenes was regulated in two ways under osmoarity: enzyme activity adjustment and gene expression response. Metabolic enzymes like PFK, PYK and G6 PDH are likely adjusted by some factors, which are regulated by osmolarity. This needs further research.