Breeding Of Kluyveromyces Marxianus Strain With High Content Of Nucleic Acid

Ribonucleic acid（RNA）,short for nucleic acid,plays an important role in gene transcription and expression and protein biosynthesis.At the same time,RNA and degradation products are also widely used in food,medicine,agriculture,and other fields.At present,RNA production methods are mainly extracted from Saccharomyces cerevisiae and Candida.However,Candida is not a food safety yeast,and Saccharomyces cerevisiae cannot use lactose naturally.Kluyveromyces marxianus is an unconventional yeast,which has the characteristics of a fast growth rate and wide metabolic substrates.At the same time,it is a biosafety（GRAS）food-grade microorganism,so it can be used in the fermentation of the food industry and the production of drugs.Whey is a by-product of cheese and casein production.At present,hundreds of millions of tons of whey are produced every year in the world,but its resource utilization is not high.Whey has high nutritional value,and its total sugar is mainly lactose,so making good use of lactose is the key to the resource utilization of whey.In this study,K.marxianus was used as the starting strain and lactose as the carbon source to explore its impact on the synthesis of RNA from milk sugar by K.marxianus through gene knockout and gene overexpression affecting the growth rate,ribosome assembly,and ribosomal DNA（r DNA）transcription.On this basis,the fermentation medium was further optimized by response surface methodology.The main research contents and results are as follows:The effect of increasing cell growth rate on nucleic acid content was explored.The engineering strains overexpressing HXT14 and HXT15 genes and knocking out CTT1,CTA1,HRP1,and PYK1 genes were constructed respectively.Compared with the original strain,the nucleic acid contents of TY20-HXT14 and TY20-?CTT1 were increased by14.88%and 8.4%respectively,and the growth rate of the recombinant strain was also slightly increased.The nucleic acid contents of HXT14 were increased by 6.28%when CTT1 was knocked out and overexpressed,but the nucleic acid contents of TY20-HXT15and TY20-?CTA1,TY20-?HRP1,and TY20-?PYK1 were not significantly changed.The r RNA expression and cell growth state of the recombinant TY20-HXT14 strain were measured.To explore the influence of increasing ribosome assembly efficiency on nucleic acid content（including BRX1,RPF1,SCH9,PKAR,FHL1,and IFH1）.Compared with the original strain,the nucleic acid content of recombinant strains TY20-BRX1,TY20-RPF1,TY20-SCH9,TY20-PKAR,TY20-FHL1,and TY20-IFH1 did not change significantly.To explore the effect of ribosomal DNA（r DNA）transcription rate on nucleic acid content.The strain TY20-?RPH1was constructed,and the strains overexpressing transcription factors TY20-RRN3 and TY20-RRN5 were constructed respectively.The fermentation results showed that the nucleic acid content of the recombinant strain was basically the same as that of the original strain compared with the original strain TY20.Taking TY20-HXT14 as the test strain and aiming at the content of intracellular ribonucleic acid（RNA）,the composition of the culture medium was optimized by a single factor experiment and response surface methodology.The optimal formula for culture medium was lactose 14%,yeast extract powder 1%,Mg SO₄0.3%,and KH₂PO₄0.2%.Under the optimized medium,the RNA content of the parental strain was 16.65%,and that of recombinant strain TY20-HXT14 was 18.37%.Compared with the recombinant TY20-HXT14 strain,the RNA content increased from 14.23%to 18.37%,29.10%higher than that before optimization.Compared with 12.43%（medium not optimized）,the RNA content increased by 47.79%.