A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies

Background: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. Results: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. Conclusions: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.open0

One-pot bio-synthesis: N-acetyl-d-neuraminic acid production by a powerful engineered whole-cell catalyst

Whole cell biocatalysis is an important tool for pharmaceutical intermediates synthesis, although it is hindered by some shortcomings, such as high cost and toxicity of inducer, mass transfer resistance caused by cell membrane and side reactions. Whole-cell catalysis using N-acetyl-d-glucosamine 2-epimerase (EC 5.1.3.8) and N-acetyl-d-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) is a promising approach for the production of Neu5Ac, a potential precursor of many anti-viral drugs. A powerful catalyst was developed by packaging the enzymes in an engineered bacterium and using a safe temperature-induced vector. Since the mass transfer resistance and the side reactions were substantially reduced, a high Neu5Ac amount (191 mM) was achieved. An efficient method was also presented, which allows one-pot synthesis of Neu5Ac with a safe and economic manner. The results highlight the promise of large-scale Neu5Ac synthesis and point at a potential of our approach as a general strategy to improve whole-cell biocatalysis

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the on-demand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application

An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies

Optimal performance of multi‐step enzymatic one‐pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two‐step one‐pot recycling cascade utilizing the thiamine diphosphate (ThDP)‐dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) and the alcohol dehydrogenase from Ralstonia sp. (RADH) for the production of the vicinal 1,2‐diol (1R,2R)‐1‐phenylpropane‐1,2‐diol (PPD) using both enzymes as catalytically active inclusion bodies (CatIBs). PfBAL is hereby used to convert benzaldehyde and acetalydehyde to (R)‐2‐hydroxy‐1‐phenylpropanone (HPP), which is subsequently converted to PPD. For recycling of the nicotinamide cofactor of the RADH, benzyl alcohol is employed as co‐substrate, which is oxidized by RADH to benzaldehyde, establishing a recycling cascade. In particular the application of the RADH, required for both the reduction of HPP and the oxidation of benzyl alcohol in the recycling cascade is challenging, since the enzyme shows deviating pH optima for reduction (pH 6–10) and oxidation (pH 10.5), while both enzymes show only low stability at pH>8. This inherent stability problem hampers the application of soluble enzymes and was here successfully addressed by employing CatIBs of PfBAL and RADH, either as single, independently mixed CatIBs, or as co‐immobilizates (Co‐CatIBs). Single CatIBs, as well as the Co‐CatIBs showed improved stability compared to the soluble, purified enzymes. After optimization of the reaction pH, the RADH/PfBAL ratio and the co‐solvent content, we could demonstrate that almost full conversion (>90%) was possible with CatIBs, while under the same conditions the soluble enzymes yielded at most >50% conversion. Our study thus provides convincing evidence that (Co‐)CatIB‐immobilizates can be used efficiently for the realization of cascade reactions, i. e. under conditions where enzyme stability is a limiting issue