Efficient Knock-out Of Tpsl And Tps2 Genes In Penicillium Chrysogenum Using Agrobacterium-mediated Transformation Method

Over the last decades,the market of ?-lactam antibiotics has tremendously increased.Penicillium chrysogenum,as the main industrial strain for the production of the(3-lactam antibiotics,has been studied extensively in the field of molecular modification and process optimization.In a large-scale penicillin fermentation process,environmental gradients(dissolved oxygen,substrate,pH,etc.)caused by mass transfer and mixing limitations will impede the further improvement of penicillin titer,rate and yield.Therefore,it is important to know how P.chrysogenum deals with these environmental gradients,which findings can serve to further improvement of the industrial penicillins and other ?-lactam antibiotics production.De Jonge et al found that in lab-scale scale-down simulators for the simulation of the substrate gradient in a large-scale penicillin fermentation,using a on-off feed regime imposed on the chemostat cultures,the intracellular amount of trehalose in a high-yielding P.chrysogenm(DS17690)can be periodically synthesized and degraded in concert with the feeding regime.This periodic turnover can give rise to a futile cycle that consumes extra ATP,which in turn influence the production of penicillin.Based on this,in this study we constructed mutant strains lacking tpsl and tps2 genes in the biosynthesis pathway of trehalose in a high-producing P.chrysogenum strain,Wisconsin 54-1255,and investigated the phenotypes of these mutant strains and the wild-type strain in batch and chemostat cultures.In this study,we successfully constructed the tpsl and tps2 knockout strains,P.chrysogenum-?tps 1 and P.chrysogenum-?tps2 using the Agrobacterium mediated transformation method,.Supervirulent Agrobacterium strain AGL-1 has been selected,the carrier is pGreen?-0179 vector,the selection marker is bleomycin and the experimental object is fresh P.chrysogenum spores.The transformation efficiency of Agrobacterium mediated transformation was up to 50%,and the efficiency of homologous recombination was up to 25%in these transformants.(Partial)blocking the trehalose pathway in P.chrysogenum can affect the formation of the spores.In PDA medium,P.chrysogenum-?tps 1 can produce a smallnumber of spores,and there are nearly no spores formation in P.chrysogenum-?tps2 strain,after 6 days of incubation on the petri dishes.Compared to the parental strain,the mycelium of P.chrysogenum-?tpsl and P.chrysogenum-?tps2 are more transparent.Unexpectedly,the glucose tolerance of the two mutants were almost the same as the Wisconsin54-1255 strain within a wide range of Cs(20 g/L to400 g/L),no morphological changes on the petri dishes were observed.In batch cultures,the maximum specific growth rate(?max,0.175h-1)of P.chrysogenum-?tpsl was similar to that of Wisconsin54-1255(0.172 h-1).The maximum biomass yield on substrate(YX/Smax)was 2.185 CmolX/molS,which was 0.59 times of Wisconsin54-1255(3.679 CmolX/molS).Under the chemostat cultivations at the dilution rate(D)of 0.05 h"1,compared to the parental strain,in P.chrysogenum-?tpsl strain,the intracellular level of the trehalose decreased and of the glucose-6-phosphate,fructose-6-phosphate and fructose-1,6-phosphate increased,the biomass specific oxygen consumption rate(qo2)can reach 1.906 mmol/gDW/h,which value was 1.1 times higher and the biomass specific rate for penicillin production(qp)was 2 times lower.The results showed that in P.chrysogenum-?tpsl strain,lacking the trehalose-6-phosphate synthase can enhance the respiratory rate.In batch cultures,as compared with the parental strain,the ?max of P.chrysogenum-?tps2 was 0.204 h-1,which was 1.19 times higher,and YX/Smax was 3.081 CmolX/molS,which was(1.2)times lower.Under chemostat cultures(D=0.05 h-1),in P.chrysogenum-?tps2,the intracellular amount of trehalose-6-phosphate reached 17times higher than that of Wisconsin54-1255,while,the intracellular amount of the trehalose was 2.4 times lower than that of Wisconsin54-1255,and the glycolytic intermediates glucose-6-phosphate,fructose-6-phosphate and fructose-1,6,-phosphate were decreased,the q02 and qco2 were 1.1 times and 1.2 times lower,respectively.The experimental results showed that the mutant lacking threhalose-6-phosphate phosphorylase had lower metabolic activity.More interesting is that the qp value increased as the cultures age and after 300 hours of the chemostat cultivation it can reach the same maximum value(1 mmol/Cmolx/h)as observed in the parental strain.