| Erythritol is a natural sweetener widely used in food,cosmetics,medicine and feed industries.The microbial fermentation method is currently the main production method of erythritol,and its production cost largely depends on the efficiency of the strain to synthesize and secrete erythritol.This paper aims to promote the synthesis and secretion of erythritol by implementing strategies such as microbial mutagenesis,enhanced fermentation process control,and improved cell permeability of strains.The main findings are as follows:(1)Mutagenic breeding and process optimization of Yarrowia lipolytica: Through the ARTP mutation breeding of the original erythritol producing strain Yarrowia lipolytica,the high erythritol producing strain Yarrowia lipolytica JZ-204 is obtained,and the yield is increased by 86.82%.On this basis,single-factor optimization and orthogonal experiment are carried out on the high-yield strain JZ-204,and the best conditions for shaking flask fermentation of erythritol are: inoculation amount 8%,initial pH 3,temperature 39?,Glucose concentration 250 g/L,liquid volume 30 mL,shaker speed 220 r/min.(2)Two-stage glucose concentration controlling strategy for erythritol production in the culture by Yarrowia lipolytica: The effect of substrate concentration on erythritol biosynthesis by Yarrowia lipolytica JZ-204 is studied on the scale of 5 L fermentor in this paper.Different initial glucose concentration is adopted in batch fermentations,and it indicated that the high glucose concentration(300 g/L and 400 g/L)contributes to the production of erythritol.In addition,it is found that the low glucose concentration(100 g/L)was beneficial to the cell growth of Yarrowia lipolytica JZ-204.Thus,a two-stage fermentation is conducted based on the control of glucose concentration.In the optimized fed-batch fermentation,the initial glucose concentration is kept at 100 g/L,and extra carbon source was added to increase the total glucose concentration to 300 g/L at 22 h.The erythritol titer obtained from the two-stage fermentation was 92.66 g/L,which was 1344.81%,84.54%,14.66% and 7.57% higher than that achieved in batch fermentations(100 g/L,200 g/L,300 g/L and 400 g/L),respectively.And the productivity of erythritol reached 0.48 g/(L·h),which was 300%,37.14%,29.73% and 20.00% higher than that obtained from batch fermentations(100 g/L,200 g/L,300 g/L and 400 g/L),respectively.This strategy significantly enhances the efficiency of erythritol synthesis by Yarrowia lipolytica JZ-204.(3)Improve the cell permeability of the strain and enhance the synthesis and secretion of erythritol: The effect of exogenous surfactant addition on the fermentation of erythritol is investigated.From comparison it is found that the effect of adding CTAB is the worst,while the effect of adding Tween 60 is the best,and the optimal dosage is 6 g/ L.The effect of the addition time of Tween 60 on the production of erythritol is studied at the shake flask level,and it is found that when Tween 60 is added at 24 h,the maximum erythritol production of 43.06 g/L could be obtained.At the same time,the fermentation time can be shortened by 14 h.On this basis,the strategy is applied to a 5L fermentor for scale-up verification.The results show that when 2 g/L Tween 60 is added,the erythritol production could reach the highest level of 88.46 g/L,which is an improvement over the control 10.82%.In order to reveal the mechanism of the effect of surfactants on cell permeability,the fatty acid composition of the cell membrane of Yarrowia lipolytica JZ-204 is measured.The results show that the addition of surfactant The addition of surfactant Tween 60 increased the content of unsaturated fatty acids in the cell membrane by 10.69%,which significantly improved the permeability of the cell membrane,made intracellular substances secrete more easily outside the cell,and weakened the product feedback suppression effect.At the same time,the decrease of extracellular nutrient uptake barrier promoted the growth of bacteria and the synthesis of erythritol. |
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