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Abstract Pyrrolysine (Pyl, O) exists in nature as the 22ndproteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes inEscherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins inE. coli.
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Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells
Abstract Improved prodrug‐activating enzymes have the potential to increase the therapeutic efficacy of gene‐directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5‐fluorocytosine (5‐FC) to the chemotherapeutic 5‐fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site‐saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants throughEscherichia coligenetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitizeE. coliand HT1080 fibrosarcoma cells to 5‐FC. Several mutants identified in this study showed increased sensitization ability for bothE. coliand HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.
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- Award ID(s):
- 1803805
- PAR ID:
- 10459566
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- AIChE Journal
- Volume:
- 66
- Issue:
- 3
- ISSN:
- 0001-1541
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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