A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6 -tagged eGFP (eGFPHis ) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases-a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)-yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.

Fe(III)-complex mediated bacterial cell surface immobilization of eGFP and enzymes.

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.

Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg-1. Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, ß-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the kcat/Km value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL-1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA.

Engineering Saccharomyces cerevisiae for Efficient Biosynthesis of Fatty Alcohols Based on Enhanced Supply of Free Fatty Acids.

In recent years, production of fatty acid derivatives has attracted much attention because of their wide range of applications in renewable oleochemicals. Microorganisms such as Saccharomyces cerevisiae provided an ideal cell factory for such chemical synthesis. In this study, an efficient strategy for the synthesis of fatty alcohols based on enhanced supply of free fatty acids (FFAs) was constructed. The FAA1 and FAA4 genes encoding two acyl-CoA synthetases in S. cerevisiae were deleted, resulting in the accumulation of FFAs with carbon chain length from C8 to C18. The coexpression of the carboxylic acid reductase gene (car) from Mycobacterium marinum and the phosphopantetheinyl transferase gene (sfp) from Bacillus subtilis successfully converted the accumulated FFAs into fatty alcohols. The concentration of the total fatty alcohols reached 24.3 mg/L, which is in agreement with that of the accumulated FFAs. To further increase the supply of FFAs, the DGAI encoding the acyl-CoA:diacylglycerol acyltransferase involved in the rate-limiting step of triacylglycerols storage was codeleted with FAA1 and FAA4, and the acyl-CoA thioesterase gene (acot) was expressed together with car and sfp, resulting in an enhanced production of fatty alcohols, the content of which increased to 31.2 mg/L. The results herein demonstrated the efficiency of the engineered pathway for the production of fatty acid derivatives using FFAs as precursors.