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Synthetic addiction extends the productive life time of engineered Escherichia coli populations.

ABSTRACT: Bio-production of chemicals is an important driver of the societal transition toward sustainability. However, fermentations with heavily engineered production organisms can be challenging to scale to industrial volumes. Such fermentations are subject to evolutionary pressures that select for a wide range of genetic variants that disrupt the biosynthetic capacity of the engineered organism. Synthetic product addiction that couples high-yield production of a desired metabolite to expression of nonconditionally essential genes could offer a solution to this problem by selectively favoring cells with biosynthetic capacity in the population without constraining the medium. We constructed such synthetic product addiction by controlling the expression of two nonconditionally essential genes with a mevalonic acid biosensor. The product-addicted production organism retained high-yield mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the nonaddicted strain completely abolished production. Using deep DNA sequencing, we find that the product-addicted populations do not accumulate genetic variants that compromise biosynthetic capacity, highlighting how synthetic networks can be designed to control genetic population heterogeneity. Such synthetic redesign of evolutionary forces with endogenous processes may be a promising concept for realizing complex cellular designs required for sustainable bio-manufacturing.

SUBMITTER: Rugbjerg P

PROVIDER: S-EPMC5877936 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Synthetic addiction extends the productive life time of engineered <i>Escherichia coli</i> populations.

Rugbjerg Peter P Sarup-Lytzen Kira K Nagy Mariann M Sommer Morten Otto Alexander MOA

Proceedings of the National Academy of Sciences of the United States of America 20180220 10

Bio-production of chemicals is an important driver of the societal transition toward sustainability. However, fermentations with heavily engineered production organisms can be challenging to scale to industrial volumes. Such fermentations are subject to evolutionary pressures that select for a wide range of genetic variants that disrupt the biosynthetic capacity of the engineered organism. Synthetic product addiction that couples high-yield production of a desired metabolite to expression of non ...[more]

PMID: 29463739

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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.

Dataset Information

Synthetic addiction extends the productive life time of engineered Escherichia coli populations.

Publications

Synthetic addiction extends the productive life time of engineered <i>Escherichia coli</i> populations.

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