Metabolic Engineering Of Aspergillus Oryzae To Produce L-malate

L-malate is an important platform chemical that has extensive applications in the food,pharmaceutical,and cosmetic industries.In recent years,biological routes have been regarded as very promising approaches as cost-effective ways to L-malate production,including enzyme conversion,or microbial fermentation.Compared with the enzyme conversion methods,microbial fermentation has superiority in more potential substrates,lower cost and more productive.Nowadays,L-malate production has made great progress via biological routes.However,in light of the existing problems at present,such as less selectivity for food safety strain,lower yields or productivities,inadequate utilization of raw materials and high concentration of other acids et al.This study used filamentous fungus Aspergillus oryzae as a producer for L-malate production.Here,we synergistically engineered the synthetic pathway in cytoplasm and L-malate transport pathway,the substrate utilization of raw corn starch,mitochondrial pathways,the pathways for byproducts synthesis and transport.Finally,the productivity of L-malate has significantly increased.Major results achieved in this study are highlighted as follows:(1)A.oryzae NRRL 3488 was used as the initial host for engineering to produce malate from glucose.Here,we achieved the overproduction of L-malate by strengthening the cytoplasmic pathway.Overexpression of pyruvate carboxylase and malate dehydrogenase genes in the rTCA pathway,improved the L-malate titer from 26.1 g/L to 42.3 g/L in shake flask culture.The oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase,increasing the L-malate titer to 58.5 g/L with a productivity of 0.65 g/L/h.(2)The export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter C4T318 from A.oryzae and an L-malate permease SpMAEI from Schizosaccharomyces pombe,improving the L-malate titer from 58.5 g/L to 89.5 g/L.We localized the transporter C4T318 to the mitochondrial membrane and matrix,respectively.Unfortunately,the mean value of dry cell weight(DCW),pellet diameter and L-malate production were significant decreased.Guided by transcription analysis of the expression profile of key genes related to L-malate synthesis,the pfk gene encoding fructokinase 6-phosphate was repressed by the feedback of high concentration metabolites.Overexpression of pfk with a strong constitutive promoter increased the L-malate titer to 93.2 g/L with a productivity of 1.17 g/L/h?(3)The strain A.oryzae MT2-P can produce 55.5 g/L L-malate directly from corn starch in shake flask culture by optimization of carbon and nitrogen sources.Overexpression of the three key amylolytic enzyme genes glaA,amyB and agdA,respectively,led to enhancive efficiency of corn starch utilization.In order to further increase the transcriptional level of glaA,five copies of a 97-bp fragment containing the cis-element of the glaA promoter(PglaA)were tandemly fused and inserted to the glaA promoter,the expression level of engineered promoter was prominently higher than the initial promoter.Co-overexpression of glaA,amyB and agdA genes with the engineered promoter resulted in the L-malate titer increased to 72.0 g/L with 100 g/L corn starch in shake flask.The L-malate titer further increased to 77 g/L by co-fermentation of 30 g/L glucose and 70 g/L corn starch.(4)According to the analysis of the metabolic kinetics of key metabolites in the cytosol and mitochondria during fermentation,malate,pyruvate,succinate,and fumarate were detected in the cytosol,whereas only malate,pyruvate,and fumarate accumulated in the mitochondria,no succinate.To reduce the concentration of fumarate,a fumarase FUMI from Saccharomyces cerevisiae,was overexpressed.The engineered strain GAAF41 could produce 88.5 g/L L-malate from 130 g/L corn starch,the concentration of succinate and fumarate decreased to 9.2 and 0.79 g/L,respectively,and the pyruvate concentration increased from 0.1~0.5 g/L to 1.2 g/L.Localization of PYC from R.oryzae(ROPYC)in the cytoplasm and mitochondria resulted in up to to 95.1 g/L malate titer.In addition,lower succinate(6.9 g/L)and fumarate(0.6 g/L)concentrations were observed,and only traces of pyruvate was detected in intracellular.Compared with improvement of the oxidative branch of the TCA cycle in the mitochondria,up-regulation of the glyoxylate bypass produced more malate(99.8 g/L),and succinate(8.3 g/L).The first step in the TCA cycle was inhibited by RNAi to weaken the oxidative TCA cycle,can increase the carbon flux to the reductive pathway.With high yield in malate titer(105.3 g/L),the concentrations of the byproducts succinate and fumarate also increased to 9.5 g/L and 1.31 g/L,respectively.(5)A dicarboxylate carrier,Sfc1 p from S.cerevisiae,which transports succinate into and fumarate out of the mitochondria,was overexpressed in the mitochondria.In the mitochondria of the derived strain PGRS,1.89 g succinate/g DCW was obtained.The malate concentration increased to 109.1 g/L,while the succinate and fumarate concentrations decreased to 6.0 g/L and 0.7 g/L,respectively.Overexpression of NOX reduced the NADH/NAD ratio within certain range,which bring about the byproduct succinate concentration of the engineered strain PGRSN-3 further reduced by 36.7%(to 3.8 g/L),meanwhile the malate titer improved to 117.2 g/L.