Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations.

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants.

Evaluating oleaginous yeasts for enhanced microbial lipid production using sweetwater as a sustainable feedstock.

Background: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH.

Rapid Cloning of Random Mutagenesis Libraries Using PTO-QuickStep.

PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli.

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production.

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures.

Adaptive Laboratory Evolution of H16 for Carbon Co-Utilization with Glycerol.

H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate.

PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries.

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3' overhangs.

Protein engineering for bioreduction of carboxylic acids.

Carboxylic acids (CAs) are widespread in Nature. A prominent example is fatty acids, a major constituent of lipids. CAs are potentially economical precursors for bio-based products such as bio-aldehydes and bio-alcohols.

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY).

Background: Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from strain PC15.

Result: After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.

An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16.

Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited.

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories.

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest.

An Efficient Transformation Method for the Bioplastic-Producing "Knallgas" Bacterium Ralstonia eutropha H16.

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic β-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis, and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism.

Production of alkenes and novel secondary products by P450 OleT using novel H O -generating fusion protein systems.

Jeotgalicoccus sp. 8456 OleT (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H O ) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleT .

Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleT.

The sp. peroxygenase cytochrome P450 OleT (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleT could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences.

Biological diversity of cytochrome P450 redox partner systems.

Cytochrome P450 enzymes (P450s or CYPs) catalyze an enormous variety of oxidative reactions in organisms from all major domains of life. Their monooxygenase activity relies on the reductive scission of molecular oxygen (O2) bound to P450 heme iron, and thus on the delivery of two electrons to the heme iron at discrete points in the catalytic cycle. Early studies suggested that P450 redox partner machinery fell into only two major classes: either the eukaryotic diflavin enzyme NADPH-cytochrome P450 oxidoreductase, or bacterial/mitochondrial NAD(P)H-ferredoxin reductase and ferredoxin partners.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam-negative P.

Polishing the craft of genetic diversity creation in directed evolution.

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed.

The role of active-site Phe87 in modulating the organic co-solvent tolerance of cytochrome P450 BM3 monooxygenase.

Understanding the effects of organic co-solvents on protein structure and function is pivotal to engineering enzymes for biotransformation in non-aqueous solvents. The effects of DMSO on the catalytic activity of cytochrome P450 BM3 have previously been investigated and the importance of Phe87 in its organic co-solvent tolerance was identified. To probe the DMSO inactivation mechanism and the functional role of Phe87 in modulating the organic co-solvent tolerance of P450 BM3, the haem domain (Thr1-Leu455) of the F87A variant was cocrystallized in the presence of 14%(v/v) and 28%(v/v) DMSO.

Directed evolution of an antitumor drug (arginine deiminase PpADI) for increased activity at physiological pH.

Arginine deiminase (ADI; EC 3.5.3.

Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR.

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly.

Directed evolution of oxygenases: screening systems, success stories and challenges.

The field of directed evolution of oxygenases (mono-, di- and epoxygenases) is rapidly advancing as an increasing number of success stories indicate. A significant number of screening systems have been developed to specifically improve oxygenase properties. Oxygenases will become very valuable biocatalysts for synthetic applications in industry when stability, cofactor and activity properties match industrial demands.