| In recent years,steroid drugs have received great attention from the biomedical industry because of their antiviral,anti-allergic and contraceptive functions.Campesterol is an important precursor for the synthesis of steroid drugs such as progesterone,17α-hydroxyprogesterone and hydrocortisone.Since it only exists in plants,the most important way to obtain it is to separate and extract it from plant tissues.（1）First,we selected the high squalene-producing strain GTy23 as the starting strain for this experiment,and used the CRISPR/Cas9 gene editing system to integrate 7-dehydrocholesterol reductase（DHCR7）from Danio rerio into the genome of Saccharomyces cerevisiae to construct the campesterol synthetic pathway.Subsequently,we eliminated the metabolic flux competition of yeast endogenous ergosterol synthesis pathway with exogenous campesterol synthesis pathway by knocking out C-22 desaturation dehydrogenase（ERG5）.The results showed that the campesterol production of recombinant Saccharomyces cerevisiae Zw503 strain was 167.91±6.00 mg·L-1.（2）In order to further dig out the more active DHCR7,we used the amino acid sequence of Dr DHCR7 as a template for bioinformatics analysis and homology comparison,and found that 75 different sources of DHCR7 such as Carassius auratus are highly homologous to Dr DHCR7.The homology is as high as 78.83%to 92.68%.Therefore,we respectively selected DHCR7 from Carassius auratus（homology 92.68%）,Cyprinus carpio（93.10%）,Triplophysa tibetana（91.37）,Labeo rohita（92.26%）,Pangasianodon hypophthalmus（82.64%）,Colossoma macropomum（86.40%）,Astyanax mexicanus（85.77%）,Cottoperca gobio（80.71%）,Xiphophorus maculatus（79.87%）with high homology to Dr DHCR7.The results suggested that the strain Zw507 integrated with Ph DHCR7 showed the highest campesterol production of 216.93±5.53 mg·L-1,which was an increase of 29.2%compared with the strain Zw503 integrated with Dr DHCR7.（3）In order to increase the expression of DHCR7 in the cell,we select 10 yeast endogenous promotors（PGK1p,GPM1p,TDH3p,TEF1p,TPI1p,GPD1p,TEF2p,ACT1p,GAL1p and TDH2p）with high-expression,which were combined with Ph DHCR7respectively.Detected by fluorescence quantitative PCR technology,it is found that the expression level of DHCR7 under the control of the promoter TEF1p is the highest,which is1.21 times the expression level under the control of the initial promoter GAL1p.The fermentation results showed that,in line with the general trend of the transcription level of DHCR7,the campesterol production of strain Zw514 with TEF1p promoter carrying DHCR7can reach 253.35±8.21 mg·L-1,which is 18.74%higher than that of strain Zw507 with GAL1p promoter carrying DHCR7.In addition,to further increase its expression level,the Ph DHCR7expression cassette was introduced repeatedly into genome of the Zw514 strain to construct a recombinant strain containing 1 to 5 copies of Ph DHCR7 expression cassette respectively.The results showed that the production of campesterol increased as the copy number of the DHCR7expression cassette increased.When the copy number was increased to 3,the campesterol production of strain Zw523 reached the highest 302.27±10.14 mg·L-1.（4）Finally,in a 5 L fermentor,the strain Zw523 was subjected to high-density fermentation using the strategy of fed-batch fermentation.The fermentation of strain Zw523 is mainly divided into two stages.In the first stage,glucose is used as the carbon source for growth,and the initial sugar concentration is 50 g·L-1.After the glucose is completely consumed,start to supplement the galactose induction medium to enter the second fermentation stage.When galactose was consumed,it was immediately supplemented to 40 g·L-1.After 144 hours of continuous fermentation,the final output of campesterol reached 916.88±11.23 mg·L-1,realizing the efficient synthesis of campesterol in Saccharomyces cerevisiae.In addition,this strain can be used as a chassis cell for subsequent biosynthesis of other steroid drugs（pregnenolone,progesterone,hydrocortisone,etc.）. |
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