Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity.As an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity.Here,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation problem.The metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia.Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein intake.TYS8500 shows great potential in future applications for PKU therapy.

Fine-tuning Bacterial Cyclic di-AMP Production for Durable Antitumor Effects Through the Activation of the STING Pathway

The stimulator of interferon genes(STING)protein is an important and promising innate immune target for tumor therapy.However,the instability of the agonists of STING and their tendency to cause systemic immune activation is a hurdle.The STING activator,cyclic di-adenosine monophosphate(CDA),produced by the modified Escherichia coli Nissle 1917,shows high antitumor activity and effectively reduces the systemic effects of the“off-target”caused by the activation of the STING pathway.In this study,we used synthetic biological approaches to optimize the translation levels of the diadenylate cyclase that catalyzes CDA synthesis in vitro.We developed 2 engineered strains,CIBT4523 and CIBT4712,for producing high levels of CDA while keeping their concentrations within a range that did not compromise the growth.Although CIBT4712 exhibited stronger induction of the STING pathway corresponding to in vitro CDA levels,it had lower antitumor activity than CIBT4523 in an allograft tumor model,which might be related to the stability of the surviving bacteria in the tumor tissue.CIBT4523 exhibited complete tumor regression,prolonged survival of mice,and rejection of rechallenged tumors,thus,offering new possibilities for more effective tumor therapy.We showed that the appropriate production of CDA in engineered bacterial strains is essential for balancing antitumor efficacy and self-toxicity.