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. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2 g L-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4 g L-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7 g L-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1 g L-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

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. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.

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.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.

Efficient degradation of various emerging pollutants by wild type and evolved fungal DyP4 peroxidases.

The accumulation of emerging pollutants in the environment remains a major concern as evidenced by the increasing number of reports citing their potential risk on environment and health. Hence, removal strategies of such pollutants remain an active area of investigation. One way through which emerging pollutants can be eliminated from the environment is by enzyme-mediated bioremediation. Enzyme-based degradation can be further enhanced via advanced protein engineering approaches. In the present study a sensitive and robust bioanalytical liquid chromatography-tandem mass spectrometry (LCMSMS)-based approach was used to investigate the ability of a fungal dye decolorizing peroxidase 4 (DyP4) and two of its evolved variants-that were previously shown to be H2O2 tolerant-to degrade a panel of 15 different emerging pollutants. Additionally, the role of a redox mediator was examined in these enzymatic degradation reactions. Our results show that three emerging pollutants (2-mercaptobenzothiazole (MBT), paracetamol, and furosemide) were efficiently degraded by DyP4. Addition of the redox mediator had a synergistic effect as it enabled complete degradation of three more emerging pollutants (methyl paraben, sulfamethoxazole and salicylic acid) and dramatically reduced the time needed for the complete degradation of MBT, paracetamol, and furosemide. Further investigation was carried out using pure MBT to study its degradation by DyP4. Five potential transformation products were generated during the enzymatic degradation of MBT, which were previously reported to be produced during different bioremediation approaches. The current study provides the first instance of the application of fungal DyP4 peroxidases in bioremediation of emerging pollutants.

Engineering Cupriavidus necator DSM 545 for the one-step conversion of starchy waste into polyhydroxyalkanoates.

Starch-rich by-products could be efficiently exploited for polyhydroxyalkanoates (PHAs) production. Unfortunately, Cupriavidus necator DSM 545, one of the most efficient PHAs producers, is not able to grow on starch. In this study, a recombinant amylolytic strain of C. necator DSM 545 was developed for the one-step PHAs production from starchy residues, such as broken rice and purple sweet potato waste. The glucodextranase G1d from Arthrobacter globiformis I42 and the α-amylase amyZ from Zunongwangia profunda SM-A87 were co-expressed into C. necator DSM 545. The recombinant C. necator DSM 545 #11, selected for its promising hydrolytic activity, produced high biomass levels with noteworthy PHAs titers: 5.78 and 3.65 g/L from broken rice and purple sweet potato waste, respectively. This is the first report on the engineering of C. necator DSM 545 for efficient amylase production and paves the way to the one-step conversion of starchy waste into PHAs.

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. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 ß-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

The chemokine lymphotactin and its recombinant variants in oral cancer cell regulation.

BACKGROUND: The expression of XCR1 receptor and its metamorphic ligand lymphotactin (hLtn) has been shown in cancers but their precise role in tumorigenesis is poorly understood including the significance of the physiologically existing hLtn monomeric (CC3) and dimeric (W55D) confirmations where the latter thought to function as the receptor antagonist. The aim of this study was to explore the functional role of bioengineered hLtn variants and the role of fibroblasts in XCR1/hLtn expression regulation in oral cancer cells (OCCL). MATERIAL AND METHODS: qRT-PCR and flow cytometry were performed to evaluate mRNA and protein expression of XCR1 and hLtn. Recombinant hLtn variants (wild-type, CC3 and W55D mutant) were designed, expressed, purified and evaluated using proliferation, adhesion and chemotaxis assays. XCR1 and hLtn expression regulation by fibroblasts was determined using indirect co-culture. XCR1 and hLtn expression in primary and metastatic OSCC tissue was assessed using immunohistochemistry. RESULTS: hLtn caused a significant decrease in OCCL XCR1 surface protein expression. hLtn CC3 mutant was highly functional facilitating proliferation and migration. Conditioned media from primary cancer-associated and senescent fibroblasts significantly upregulated XCR1 and hLtn mRNA expression in OCCL. Immunohistochemistry revealed higher XCR1 and hLtn expression in metastatic tumour deposits and surrounding stroma compared to primary OSCC tissue. CONCLUSIONS: The development of hLtn biological mutants, regulation of XCR1 expression by its ligand hLtn and crosstalk with fibroblasts are novel findings suggesting an important role for the XCR1/hLtn axis within the OSCC tumour microenvironment. These discoveries build upon previous studies and suggest that the hLtn/XCR1 axis has a significant role in stromal crosstalk and OSCC progression.

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

Cupriavidus necator 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, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate-glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

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. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.

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. However, carboxylate reduction is a challenging chemical transformation due to the thermodynamic stability of carboxylate. Carboxylic acid reductases (CARs), found in bacteria and fungi, offer a good solution to this challenge. These enzymes catalyse the NADPH- and ATP-dependent reduction of aliphatic and aromatic CAs. This review summarised all the protein engineering work that has been done on these versatile biocatalysts to date. The intricate catalytic mechanism and structure of CARs prompted us to first examine their domain architecture to facilitate the subsequent discussion of various protein engineering strategies. This then led to a survey of assays to detect aldehyde formation and to monitor aldenylation activity. Strategies for NADPH and ATP regeneration were also incorporated, as they are deemed vital to developing preparative-scale biocatalytic process and high-throughput screening systems. The objectives of the review are to consolidate CAR engineering research, stimulate interest, discussion or debate, and advance the field of bioreduction.

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 Escherichia coli 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 Pleurotus ostreatus 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 (2.7-fold improvement in k cat/K m) and higher H2O2 tolerance (sevenfold improvement based on IC50). CONCLUSION: To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.

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. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., P phaC1, P rrsC, P j5, and P g25) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible P BAD promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of P BAD promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli.

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. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds.

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 ß-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. To overcome this limitation, a systematic study is performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells are made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieves a transformation efficiency of (3.86 ± 0.29) × 105 cfu µg-1 DNA, a 103 -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism.

Rapid Construction of Recombinant Plasmids by QuickStep-Cloning.

QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.

QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids.

BACKGROUND: Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. RESULT: Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3'-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. CONCLUSION: QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists.

The Mutagenesis Assistant Program.

Mutagenesis Assistant Program (MAP) is a web-based statistical tool to develop directed evolution strategies by investigating the consequences at the amino acid level of the mutational biases of random mutagenesis methods on any given gene. The latest development of the program, the MAP(2.0)3D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein. The combined information can be used to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants. Hence, the MAP(2.0)3D server facilitates the "in silico" prescreening of the target gene by predicting the amino acid diversity generated in a random mutagenesis library. Here, we describe the features of MAP(2.0)3D server by analyzing, as an example, the cytochrome P450BM3 monooxygenase (CYP102A1). The MAP(2.0)3D server is available publicly at http://map.jacobs-university.de/map3d.html.

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. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.