Study On The Construction Of Engineering Strain Of Candida Tropicalis Producing 10-Hydroxy Decanoic Acid

Due to hydroxy fatty acid contains both hydroxyl and carboxyl groups,it is a fatty acid with great application value and has great development prospects in high-performance materials,foods,emulsifiers and medical treatments.Among many hydroxy fatty acids,10-hydroxy decanoic acid stands out because its hydroxyl and carboxyl groups are located at both ends of the carbon chain.It can provide better performance for high-performance materials and is also an important intermediate for the synthesis of some important health-care substances.At present,the yield of chemically synthesized 10-hydroxy decanoic acid is not high,which limits its application.If 10-hydroxy decanoic acid can be synthesized in microorganisms,it will bring huge economic benefits.As a microorganism capable of utilizing alkanes and oils,Candida tropicalis(C.tropicalis)provides the innate conditions for the production of 10-hydroxy decanoic acid due to its strong ?-oxidation pathway.This study developed the C.tropicalis 1798 gene traceless editing technique to achieve efficient knock the target gene of the strain out and studied the role of four fatty alcohol dehydrogenases in the synthesis of 10-hydroxy decanoic acid in C.tropicalis 1798,which laid a solid foundation for the better development of C.tropicalis and the production of 10-hydroxy decanoic acid in the future.The main researchs and results are as follows:(1)This method relied on single plasmid traceless knockout system,used Cas9 as a positive and reverse screening marker,played a role under the induction of galactose,through screening.A double-copy traceless knockout strain of adh2 and adh3 was obtained.This study explored and constructed a set of methods based on Cas9 to efficiently edit C.tropicalis 1798 gene,trying different schemes during the construction process and finally obtaining a set of editing methods capable of editing C.tropicalis 1798 gene.Based on the single plasmid traceless knockout system,Cas9 was used as the forward and reverse screening marker under the induction of galactose to obtain a double-copy traceless knockout strain of adb2 and adh3 by screening.(2)Four key fatty alcohol dehydrogenases from the oxidative pathway of C.tropicalis 1798 were knocked out using different knockout methods,and the effects of each knocked out fatty alcohol dehydrogenase on the production of hydroxy fatty acids from the metabolic fatty acids of C.tropicalis 1798 were verified and analyzed.When the single-copy gene of adh1 was knocked out,the amount of 10-hydroxy decanoic acid reached 34.13 mg/L.When the single-copy gene of adh4 was knocked out,the amount of 10-hydroxy decanoic acid reached 58.43 mg/L.When the double copies of adh2 and adh3 were knocked out,the amount of 10-hydroxy decanoic acid reached 184.17 mg/L.(3)When knocking out the single-copy fatty alcohol dehydrogenase adh1,adh4,and double-copy fatty alcohol dehydrogenase adh2 and adh3 gene engineering bacteria,optimal fermentation conditions were 30 ?,220 rpm/min,and 10% substrate concentration.(4)After different fatty alcohol dehydrogenase were knocked out,the effect of knockout on the strain was determined by fermentation experiments.The results showed that when the single-copy genes of adh1 and adh4 were knocked out,the growth status of the strain was not affected.But when the double-copy genes of adh2 and adh3 were knocked out,the growth of the strain was lagging with no enormous negative effects.