# A Review of the Use of Native and Engineered Probiotics for Colorectal Cancer Therapy
## 3 Genetically Modified-Bacteria for Colorectal Cancer (CRC) Treatment
### Engineered Bacteria Treat CRC via Therapeutic Protein Delivery
#### 3.1.2 Prodrug-Converting Enzymes  
The targeted delivery of prodrug-converting enzymes with engineered bacteria is a therapeutic strategy for converting prodrugs into cytotoxic products at the tumor site. It aims to yield effective concentrations of anticancer drugs in the tumor microenvironment (TME) along with minimized adverse effects on organisms. Cytosine deaminase (CD), produced by bacteria and fungi, is a kind of representative prodrug-converting enzyme. CD can convert 5-fluorocytosine (5-FC), a non-toxic prodrug, into 5-fluorouracilacil (5-FU), a toxic chemotherapeutic agent [66]. A study was designed to develop an engineered attenuated strain of _S. typhimurium_ expressing CD from _E. coli_. In an MC38 tumor-bearing C57 BL/6 mouse model, this engineer accumulated in tumors at levels 1000-fold higher than those in normal tissues after a single intravenous bolus administration. High levels of 5-FU were detected in tumor tissues when MC38 mice were treated with this engineered strain and 5-FC. In addition, an anti-tumor effect was observed in MC38 mice treated with both engineered bacteria alone and engineered bacteria in combination with 5-FC but not in mice treated with 5-FC alone [67]. Interestingly, wild-type probiotic EcN is inherently capable of activating several prodrugs including CB1954, 5-FC, AQ4N, and Fludarabine phosphate, and resisting their toxicity. The combination administration of EcN and prodrugs showed a significant antitumor effect in BALB/c mice bearing CT26 tumors [68]. Even though this activation mechanism of EcN has not been elucidated, it serves as a natural enzyme pool that will facilitate the development of more prodrug-converting enzymes.  
Given the higher nutrient demand for amino acids in cancer cells, it is probably an efficient cancer therapeutic strategy to employ bacteria to deliver specific amino-acid-depletion enzymes for the deprivation of essential amino acids in the TME. Asparaginase (L-ASNase) of _E. coli_ origin is an anti-cancer protein for acute lymphoblastic leukemia [69]. It converts asparagine into aspartate and glutamine into glutamate at a low level. Some cancer cell types have no or little asparagine synthetase expression, so they cannot convert aspartate back into asparagine for further use [70]. In this case, cancer cells will suffer from asparagine starvation and subsequently experience apoptosis. [PERSON] et al. engineered attenuated _S. typhimurium_ to produce L-ASNase to counter tumors. This strain expressed L-ASNase under the control of the inducible araBAD promoter system for the control of gene-selective expression within solid tumors. As a result, the L-ASNase expressed and secreted by _S. typhimurium_ induced apoptotic cancer cells in vitro, and this engineered strain significantly promoted tumor regression and increased survival in an MC38 mouse model [71].  
Another strategy is to utilize a dietary prebiotic as a prodrug and convert it into anticancer compounds using specific enzymes. One recent study engineered EcN to secrete myrosinase, an enzyme that converts dietary glucosinolates, naturally occurring components of cruciferous vegetables, into the anti-cancer molecule sulforaphene (Figure 3). This strategy succeeded in killing over 95% of human and murine CRC cell lines in vitro and reducing over 75% of tumors in azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice [20; 72].  
Figure 2: Engineered bacteria harboring cytolysin A protein (ClyA, a pore-forming cytotoxin) can act on tumor cells by forming transmembrane pores and inducing cell apoptosis.