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Total synthesis of Escherichia coli with a recoded genome.

ABSTRACT: Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

SUBMITTER: Fredens J

PROVIDER: S-EPMC7039709 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Total synthesis of Escherichia coli with a recoded genome.

Fredens Julius J Wang Kaihang K de la Torre Daniel D Funke Louise F H LFH Robertson Wesley E WE Christova Yonka Y Chia Tiongsun T Schmied Wolfgang H WH Dunkelmann Daniel L DL Beránek Václav V Uttamapinant Chayasith C Llamazares Andres Gonzalez AG Elliott Thomas S TS Chin Jason W JW

Nature 20190515 7757

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase s ...[more]

PMID: 31092918

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Project description:BackgroundHydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource.ResultsWe synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D.ConclusionsUsing different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.

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Total synthesis of Escherichia coli with a recoded genome.

Publications

Total synthesis of Escherichia coli with a recoded genome.

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