Fatty Acids Production By Leucine Mutant Ogataea Polymorpha

Fatty acids,as an important feedstock of oleochemical,have been received much attention.However,natural fatty acids are routinely generated from animal fats and vegetable oils,which can not meet the expanding demand in market.Therefore,the development of new sources of fatty acids has become a research hotspot.Ogataea polymorpha has been recognized as a robust microbial cell factory for the production of fatty acids featuring minimal limitation by environment and region,and short production cycle.However,O.polymorpha belongs to a leucine mutant.Leucine can not be synthesized due to the mutation of LEU2 gene.In order to make full use of the potential of the microbial cell factory to produce fatty acids and realize industrial success,the following studies were carried out:It was found that leucine was very important for the growth of O.polymorpha.When the leucine concentration was lower than 200 mg/L,the strain was very unstable.The strain randomly exhibted as nearly no cell growth,slow growth,or normal growth.When the concentration of leucine exceeded 200 mg/L,the growth of the strain became stable with the increase of leucine concentration.However,the yield of fatty acid showed the opposite trend.It was indicated that the leucine supplementation strategy was not facilitated for the fermentation control.The fatty acids produced by O.polymorpha were mainly composed of long-chain fatty acids with 16 and 18 carbon atoms.Specially,palmitic acid（c16:0）,oleic acid（C18:1）and linoleic acid（C18:2）together accounted for over 90%the total fatty acids.The expression of LEU2 gene was found beneficial for the growth stability of O.polymorpha,regardless of the expression methods.Among them,the recombinant strain FD09 which expressing Op LEU2 in situ was the most stable.It was suggested that FD09could synthesize a large amount of leucine to meet the requirment through a self-regulation mechanism which could coordinate the growth and fatty acid synthesis of the strain.Addition of leucine exerted on effect on the cell growth and fatty acid production of the FD09.The culture conditions of fatty acids production by FD09 were optimized.The highest fatty acid titer reached 1.86 g/L when the C/N ratio,initial p H,culture temperature and OD_600nmof seed culture were set at 120,6.4,37℃and 6-8,respectively,in shake flask.This data was dramatically increased to 18 g/L when FD09 was incubated in 1-L stirred-tank fermentor through the fed-batch fermentation.The C/N ratios of the fermentation medium and the feeding medium were set at 17.5 and 120,respectively.The agitation and aeration rate were set at 800 rpm and 36 s L/h,respectively.A dissolved oxygen（DO）control feeding strategy was performed.Specially,the feeding was initiated when DO was higher than 30%and terminated when DO was lower than 30%.This study verified the importance of endogenous expression of leucine on cell growth and fatty acid production,provided technical support for the commerical success of fatty acids production by O.polymorpha,and could served as an important reference for the application of microbial cell factory.