| β-carotene is a fat-soluble natural pigment with the the color of orange.In humansβ-carotene,as a typical member in the carotenoids family,is transformed into vitamin A,so that is being called the precursor of vitamin A.It may physiologically function as an antioxidant,has properties protective against cancer,stimulates the immune system and others.Currently,natural β-carotene,as a high quality food colorant and nutritional supplement,is widely being used in food,feed and health products.However,the output of natural β-carotene is too low,which limits its application.This study aimed to promot the P-carotene synthesis in recombinant Saccharomyces cerevisiae though inhibiting the metabolic bypass around β-carotene biosynthesis pathway to increase the supply of precursor materials.While made the recombinant cells at later growth stage starved of nitrogen to evaluate the possibility of improving β-carotene production under this condition.The main research contents are:1.Reduce ethanol production to improve β-carotene production and the sustainability of fermentationADH1,the key gene in ethanol synthesis,was deleted from the original strain(W)which was used to be integrated with β-carotene synthesis genes,This deletion increased the cell diameter by 0.31 times,When ADH1 was deleted from the strain already integrated with P-carotene synthesis genes(C),to obtain an engineering strain(C-A1),the cell size did not change with deletion.When ethanol content was detected with HSGC-FID,it was found that expression of β-carotene biosynthesis-related genes in Saccharomyces cerevisiae enhanced the ethanol production by 9.8%at 4.03 mg/mL.The deletion of ADH1 in C decreased ethanol production by 50.1%compared with C.At the later fermentation stage of day 5,the color of C-A1 cells turned to orange.What’s more,compared with C,the dots of carotenoid fluorescence in C-Al cells increased and became brighter.When fermented in YPD,the yield of β-carotene in C-A1 cells increased by 17.0%in compared with C,was up to 2.98 μg/mg DCW(dry weight),and had no significant change in the production of lycopene,which was about 1.40 μg/mg DCW.However,with fermentation in YPD at day 7,lycopene could not be detected.2.Reduce the ergosterol content to increase the supply of precursor materials toβ-carotene synthesisDeleted the nonessential genes(ERG3,ERG4 and ERG5)in the ergosterol synthesis pathway from C to construct three engineering strains,C-E3,C-E4 and C-E5.Among them,the growth of C-E3 was strongly impaired at low temperature,while the other two grew well.Compared with C,the cell diameters of C-E3 and C-E4 were increased by 0.20 and 0.15 folds.With HPLC detection for ergosterol content,it was found that the expression ofβ-carotene biosynthesis-related genes in Sacharomyces cerevisiae increased the ergosterol production by 14.6%at 11.54 μg/mg DCW,As expected,there is almost no ergosterol in three engineering strains which had been knocked out ergosterol synthesis-related genes,and the yield of β-carotene is different among them.Fermentation in YPD,the highest yield of β-carotene in C-E3 is 2.74 μg/mg DCW,which increased by 7.2%compared with C.Theβ-carotene content in C-E4 and C-E5 cells was not much,the highest yields in which were 2.21 μg/mg DCW and 1.85 μg/mg DCW,respectively.Compared with C,reduced by 16.9%and 27.9%,but total carotenoids were all increased.Among ERG3,ERG4 and ERG5,as only deletion of ERG3 could reduce the ergosterol synthesis and increase β-carotene production,ERG3 was selected to delete together with ADHl.As a direct deletion of both genes with a regular way failed,ERG3 in a URA3 plasmid was complemented to C-E3 cells to delete ADH1 by a conventional method.After that,the plasmid wsa removed to obtain an engineering strain C-E3A1.Like C-E3,the growth of C-E3A1 was significantly impaired at low temperature,the diameters of its cells were 1,50 times of C or C-A1,and were about 0.27 times than C-E3.The color of C-E3A1 cells in the later fermentation stage is orange,and dots of carotenoid fluorescence were densely distributing throughout the cell.Fermentated in YPD,the P-carotene yield in C-E3A1 was up to 3.75 μg/mg DCW,increased by 40.7%,46.3%and 28.9%compared with C and engineering strains C-E3 and C-A1.Yield of total carotenoids were 2.08 times of C,increased by 37.7%and 90.8%compared with C-E3 and C-A1.3.Nitrogen starvation promote P-carotene biosynthesisThe microbial fermentation products were affected not only by genes regulation,but also by growing conditions.It was reported that yeast cells produced more lipid droplets and ergosterol under nitrogen starvation at the later fermentation stage.Our data showed that under nitrogen starvation,ergosterol content was up to 17.16 μg/mg DCW in W,an increase of 34.0%compared with growth in YPD for the same period.While in the cells of C and C-Al,ergosterol contents were only 13.93 μg/mg DCW and 12.19 μg/mg DCW,increased by only 3.1%and 6.9%compared with fermentation in YPD for the same period,but lower than W by 18.8%and 27.8%.Expression of P-carotene biosynthesis-related genes in Saccharomyces cerevisiae to increased the ergosterol production in cells cultured in YPD,but ergosterol synthesis could be partly inhibited by shifting the cells to nitrogen starvation conditions at the later fermentation stage,This switch could,possibly prepare the substances for the more efficient synthesis of P-carotene.With nitrogen starvation,the color of cells producing β-carotene became darker,and the intensity of carotenoid fluorescence dots were enhanced.Under this conditionβ-carotene production of C was up to 4.49 μg/mg DCW,with an increase of 68.2%compared with fermentation in YPD for the same period,and lycopene was not detected.For C-E3,the yield of β-carotene was 3.59 μg/mg DCW,and an increase of 40.2%.For C-E3A1,with an increase of 32.0%,produced 5.07 μg/mg DCW of β-carotene.C-A1 had the highest increase of 92.1%,reached to 5.59 μg/mg DCW,compared with fermentation in YPD for the same period.As β-carotene carriers,lipid droplets increased alone would not affect P-carotene synthesis much.However,under nitrogen starvation,both β-carotene production and lipid droplets accumulation inceased simultaneously,which may benefit the the effective production of β-carotene. |
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