| Erythritol, 1,2,3,4-butantetraol, is widely applied in the food and pharmaceutical industries as a biological sweetener. It has many advantages just like low moisture absorption, better thermostability, anticarious. Currently, erythritol is fermented by osmophilic yeast using sucrose, glucose and enzyme hydrolyzed starch grains as carbon source, the cost of raw materials is high and generating food security problem. Glycerol serving as a raw material have been widely used in various biotechnological processes for valuable products fermentation. As high osmotic pressure with glycerol, benefiting to high yield of erythritol, but the inhibition effect on cell growth leading to the low biomass and erythritol productivity, which is the key problem in such process. In this study, some hyperosmotic mutants of Yarrowia lipolytica were obtained through high-efficiency mutagenesis and screening method. The low-cost hyperosmotic medium was optimizated and the fermentation kinetics of erythritol production was established. A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production was developed. And the osmotic stress response mechanisms of Y. lipolytica were explained at proteomics level and transcriptional level. The main results and conclusions are summarized as follows:(1) Mutation screening of hyperosmotic yeast Y. lipolytica with high erythritol production from glycerolY. lipolytica CICC 1675 using glucose as carbon source was multiple-mutated by ARTP and DES, and high-efficiency visual TTC hyperosmotic medium with glycerol as a sole carbon source was applied to screen the hyperosmotic mutants. Y. lipolytica Y-07 was successfully isolated from 500 mutants which was better erythritol fermentation performance with glycerol. The biomass was 12.8 g·L–1,the production of erythritol achieved 43.5 g·L–1 with 0.36 g·g–1 yield, which were improved 91.0%, 217.5%, 71,4% more than the original strain. And Y-07 was with a high stability of genetic traits. The medium composition and culture conditions were preliminarily studied and determined as follows: glycerol 200 g·L–1, urea 1.68 g·L–1, yeast extract 0.5 g·L–1, corn steep liquor 0.5 g·L–1, CaCl2·2H2O 500 mg·L–1, ZnSO4·7H2O 100 mg·L–1, MnSO4·4H2O 10 mg·L–1, FeSO4·7H2O 100 mg·L–1, MgSO4·7H2O 500 mg·L–1, KH2PO4 0.2 g·L–1, pH 3.0, inoculom age 24 h, inoculom size 10%(v·v–1), culture volume 20/250 mL. Erythritol concentration was 69.4 g·L–1, yield(Yp/s) was 0.37 g·g–1, productivity(P) was 0.41 g·L–1·h–1, which were improved 263.4%, 60.9%, 272.7% more than the original strain under optimal cultrure conditions.(2) Optimization of hyperosmotic medium and establishing the fermentation kinetics of erythritol production by Y. lipolytica from glycerolGlycerol, urea and NaCl relating to osmotic pressure were determined as the most significant factors affecting erythritol production by Plackett-Burman design. A CCD experiment was designed with 3 factors and 5 levels, and RSM and ANN models were established using the data of CCD to search the optimal compositions of the significant factors. The fitting accuracy of ANN model was more accurate compared to RSM. The optimal solution was searched by GA, the predictive maximum erythritol production was 110.7 g·L–1 and the corresponding optimum input conditions were found as glycerol 232.39 g·L–1, urea 1.57 g·L–1, NaCl 31.03 g·L–1. And the experimental maximum erythritol concentration reached 109.2 g·L–1. Erythritol fermentation kinetics was investigated in a batch system. Multi-step fermentation kinetic models with hyperosmotic inhibitory effects were developed. The resulting mathematical equations provided good description of temporal variations such as microbial growth(X), substrate consumption(S) and product formation(P) in erythritol fermentation. The correlation coefficients of the kinetic parameters mentioned above were 0.981, 0.994, and 0.992, respectively indicating that the model predictions were highly agreement with the experimental data. The accordingly derived model provided a visual description for the erythritol batch fermentation process, and a kinetic theoretical basis for osmotic pressure control fed-batch fermentation.(3) Two-step osmotic pressure control fed-batch fermentation strategy for improvement of erythritol productionIt was found that polyols production was definitely influenced by osmotic pressure conditions. Polyols yield and rE/M elevated with osmotic pressure increasing. Erythritol production increased more significantly. Mannitol was inhibited drastically with NaCl addition. However, the yeast growth was inhibited. The optimal osmotic pressure for erythritol production was 4.17 osmol·kg–1. The specific activity of ER was elevated at high osmotic pressure(4.17 osmol·kg–1), which was almost twice higher than that at low osmotic pressure(3.21 osmol·kg–1). However, the specific activity of M-1-PDH exhibited the opposite trend. A two-stage osmotic pressure control fed-batch strategy based on the kinetic analysis was developed for higher erythritol yield and productivity. Glycerol was fed not noly as substrate but also osmotic regulator. The osmotic pressure was maintained at 4.25 osmol/kg by feeding glycerol in cell growth phase. After 132 h, the osmotic pressure was controlled at 4.94 osmol/kg to maintain a high dpery/dt. Maximum erythritol production of 194.3 g/L was obtained with 0.95 g·L–1·h–1 productivity, which were 25.7% and 2.2% improvement over the best results in one-stage fed-batch fermentation, respectively. This is the highest reported value of erythritol production from glycerol up to now. High production, high yield and high productivity of erythritol were achieved simultaneously under high osmotic pressure.(4) The study on osmotic stress response mechanisms of Y. lipolytica at proteomics level and central metabolic genes transcriptional levelProteomic analyses allowed identification of 44 differentially expressed proteins among the proteins distributed in the range of pI 3-10 and molecular weight 14.4-97.4 kDa between the osmotic stress conditions. Remarkably, the main proteins were involved in the pathway of energy, metabolism, cell rescue and stress response. The improvement of erythritol production under high osmotic stress was due to the significantly induced of a range of crucial enzymes related to polyols biosynthesis, such as TKL, TPI and AKRs family. Additionally, the high osmotic stress could also induce the other cell stress responses as with heat shock and oxidation stress responses, and these responsive proteins such as HSPs family, CAT and SOD were also increased the expression levels drastically under hyperosmotic pressure. The expression of such enzymes related to protein and nucleotide biosynthesis were inhibited drastically reflecting the growth arrest of Y. lipolytica under hyperosmotic stress, such as 60 S ribosomal protein L2/L4, Translation elongation factor, phosphoribosyl transferase. RT-PCR was applied to analysis the transcriptional levels of the genes involved in central metabolic pathway, erythritol synthesis, oxidation stress related proteins. The results suggested that high osmotic pressure could inhibit the transcriptional levels of the genes in EMP pathway just like TDH1, PGI1, PFK1, PYK1. The genes, GUT1, FBP1 involved in gluconeogenesis pathway, and CIT1, KGD1 involved in TCA circle were induced under high osmotic pressure, which enhanced the efficiency of substrate ultilization and energy supplement. Additionally, the transcriptional levels of the genes TKL1, ER1 related to erythritol biosynthesis in HMP pathway were also increased. And the transcriptional levels of SOD1, CAT1 belonging to oxidation stress response genes were also induced drastically. The reason of erythritol improvement under high osmotic pressure might be metabolic flux deflection from EMP to HMP.(5) Improvement of erythritol productivity with osmoprotectant and antioxidant additionThe composition and concentration of intracellular free amino acids were affected by different initial osmotic pressure. High osmotic pressure elevated the total concentration of intracellular free amino acids. Glycine and proline were excellent antioxidant, Y. lipolytica could import extracellular amino acid and accumulate as a compatible solute in cytoplasm in order to resist the damage of the high osmotic pressure. The specific activities of SOD and CAT were increased under high osmotic pressure, but which were reduced appropriately with antioxidants(Asc, Cys) addition. Osmoprotectant and antioxidant could reduce the period of erythritol fermentation, increase glycerol consumption rate and erythritol productivity. When 30 mg·L–1 glycine, 40 mg·L–1 proline, 30 mmol·L–1 Asc and 3.0 mmol·L–1 Cys were added at the beginning of the fermentation, the fermentation period was reduced to 174 h, and the glycerol consumption rate was 2.41 g·L–1·h–1, erythritol productivity were 1.13 g·L–1·h–1, which had increased-14.7%, 23.6% and 18.9%, respectively. Erythritol productivity was further improved. The final concentration and yield of erythritol were not affected remarkably. |
