Transporter Engineering In Yarrowia Lipolytica For High-efficient Succinic Acid Production

Succinic acid is an important intermediate of organic synthesis.The global annual output of succinic acid is 70-90 kilotons,which is widely used in food,agriculture,chemical and other fields.Succinic acid can be used as intermediates or important precursors to synthesize important compounds such as y-butyrolactone,tetrahydrofuran and biodegradable plasti.Therefore,the efficient production of bio-succinate has gradually become a research hotspot in recent years.Yarrowia lipolytica is a very important industrial microorganism.Like Pichia pastoris,Kluyveromyces lactis and Hansenula polymorpha,it is an unconventional yeast.It has unique physiological and metabolic characteristics such as metabolism of oil and strong stress resistance.In recent years,with the rapid development of synthetic biology and gene editing technology,scientists have successfully constructed a Yarrowia lipolytica cell factory with high value-added industrial products,including biodiesel,recombinant proteins,organic acids,lipids and their derivatives and fuel ethanol,which makes this yeast has great application potential in food,medicine and biochemical energy.In particular,Y.lipolytica has a strong ability to metabolize carbon in the tricarboxylic acid cycle center and could also tolerate lower pH conditions,so it is an ideal platform for the production of succinic acidAt present,however,the production of succinic acid is still low in the medium with glucose as the sole carbon source.In order to solve this problem,this study started from two aspects:the optimization of transport system and the rational transformation of metabolic pathway,so as to realize the efficient production and secretion of succinic acid from glucose.First of all,we screened and characterized five mitochondria carboxylic acid carriers related to the transport of carboxylic acid,and found that YlDic protein may promote the transport and secretion of succinic acid.Then,we characterized the membrane-anchored dicarboxylic acid transporters from nine different fungi and found that SpMaelp from Schizosaccharomyces pombe is the key transporter and secreted by succinic acid.In addition,we built a chassis cell with better acidogenic performance by overexpressing the key enzymes in the three metabolic pathways related to succinic acid production-oxidized TCA cycle,reduced TCA cycle and glyoxylic acid cycle.Finally,we integrated the optimization of transport system and metabolic pathway,and constructed an engineering strain PGC62-SYF-Mae with strong succinic acid production capacity with better growth.In the process of fed-batch fermentation,the strain produced succinic acid 101.4 g/L in the bioreactor,the productivity was 0.70 g/L/h,and the glucose conversion rate was 0.37 g/g.At present,the succinic acid yield of this strain is the highest among the yeasts with glucose as the sole carbon source.Our results show that transporter engineering is an effective strategy for Y.lipolytica to secrete succinic acid efficiently and will further promote the industrial production of biosuccinic acid.