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Kunjapur, Aditya

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Kunjapur

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Aditya

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Kunjapur, Aditya
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    Engineering posttranslational proofreading to discriminate nonstandard amino acids
    (National Academy of Sciences, 2018) Kunjapur, Aditya; Stork, Devon; Kuru, Erkin; Vargas-Rodriguez, Oscar; Landon, Matthieu; Söll, Dieter; Church, George
    Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called “posttranslational proofreading” to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.
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    Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway
    (BioMed Central, 2016) Kunjapur, Aditya; Hyun, Jason C.; Prather, Kristala L. J.
    Background: Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin. Results: Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and l-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-l-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33 % (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25 % increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability. Conclusions: Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or methionine biosynthesis, we pursued two orthogonal strategies that are each aimed at deregulating multiple reactions. Our results increase the working knowledge of SAM biosynthesis engineering and provide a framework for improving titers of metabolic products dependent upon methylation reactions. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0459-x) contains supplementary material, which is available to authorized users.