Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime.

Naturally occurring and engineered flavin-binding, blue-light-sensing, light, oxygen, voltage (LOV) photoreceptor domains have been used widely to design fluorescent reporters, optogenetic tools, and photosensitizers for the visualization and control of biological processes. In addition, natural LOV photoreceptors with engineered properties were recently employed for optimizing plant biomass production in the framework of a plant-based bioeconomy. Here, the understanding and fine-tuning of LOV photoreceptor (kinetic) properties is instrumental for application. In response to blue-light illumination, LOV domains undergo a cascade of photophysical and photochemical events that yield a transient covalent FMN-cysteine adduct, allowing for signaling. The rate-limiting step of the LOV photocycle is the dark-recovery process, which involves adduct scission and can take between seconds and days. Rational engineering of LOV domains with fine-tuned dark recovery has been challenging due to the lack of a mechanistic model, the long time scale of the process, which hampers atomistic simulations, and a gigantic protein sequence space covering known mutations (combinatorial challenge). To address these issues, we used machine learning (ML) trained on scarce literature data and iteratively generated and implemented experimental data to design LOV variants with faster and slower dark recovery. Over the three prediction-validation cycles, LOV domain variants were successfully predicted, whose adduct-state lifetimes spanned 7 orders of magnitude, yielding optimized tools for synthetic (opto)biology. In summary, our results demonstrate ML as a viable method to guide the design of proteins even with limited experimental data and when no mechanistic model of the underlying physical principles is available.

Design, Production, and Characterization of Catalytically Active Inclusion Bodies.

Catalytically active inclusion bodies (CatIBs) are promising biologically produced enzyme/protein immobilizates for application in biocatalysis, synthetic chemistry, and biomedicine. CatIB formation is commonly induced by fusion of suitable aggregation-inducing tags to a given target protein. Heterologous production of the fusion protein in turn yields CatIBs. This chapter presents the methodology needed to design, produce, and characterize CatIBs.

Catalytically Active Inclusion Bodies─Benchmarking and Application in Flow Chemistry.

In industries, enzymes are often immobilized to obtain stable preparations that can be utilized in batch and flow processes. In contrast to traditional immobilization methods that rely on carrier binding, various immobilization strategies have been recently presented that enable the simultaneous production and in vivo immobilization of enzymes. Catalytically active inclusion bodies (CatIBs) are a promising example for such in vivo enzyme immobilizates. CatIB formation is commonly induced by fusion of aggregation-inducing tags, and numerous tags, ranging from small synthetic peptides to protein domains or whole proteins, have been successfully used. However, since these systems have been characterized by different groups employing different methods, a direct comparison remains difficult, which prompted us to benchmark different CatIB-formation-inducing tags and fusion strategies. Our study highlights that important CatIB properties like yield, activity, and stability are strongly influenced by tag selection and fusion strategy. Optimization enabled us to obtain alcohol dehydrogenase CatIBs with superior activity and stability, which were subsequently applied for the first time in a flow synthesis approach. Our study highlights the potential of CatIB-based immobilizates, while at the same time demonstrating the robust use of CatIBs in flow chemistry.

Construction and comprehensive characterization of an EcLDCc-CatIB set-varying linkers and aggregation inducing tags.

BACKGROUND: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. RESULTS: Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L- 1 d- 1 and a specific volumetric productivity of 256 g L- 1 d- 1 gCatIB-1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L- 1 d- 1, specific volumetric activity: 106 g L- 1 d- 1 gCatIB- 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. CONCLUSIONS: Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.

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. KEY POINTS: • Catalytically active inclusion bodies (CatIBs) are promising bionanomaterials. • Potential applications in biocatalysis, synthetic chemistry, and biotechnology. • CatIB formation represents a generic approach for enzyme immobilization. • CatIB formation efficiency depends on construct design and expression conditions.