Non-native Enzymes Research Articles

Designing Microbial Cell Factories Through the Application of Metabolic Engineering

Concepts and Applications of Metabolic Engineering for Metabolites Production Engineering: Open Access New avenues for a deeper knowledge of the organism's physiology and metabolism have been made possible with the advancement in molecular biology tools and access to the whole genome sequencing data that open the doors for metabolic engineers and sped up the metabolic engineering application. It is pertinent to say that without molecular biology which has numerous applications in modern biotechnology, metabolic engineering couldn’t be in its present bloomy position. Metabolic engineering permits the introduction of novel, beneficial features such as drought and salt tolerance in plant biotechnology, assisting the discovery of disease-causing genes and their therapy in medical biotechnology. Nikel & de Lorenzo, (2021) accentuated the use of metabolic engineering for the degradation of recalcitrant compounds in environmental biotechnology. Metabolic engineering can fabricate the coveted metabolite using renewable resources by altering the endogenous pathways or employing the heterologous biosynthetic pathways in microbes. Thus, microbes perform as a cell factory for producing different metabolites using several native and non-native enzymes. Due to this, metabolic engineers and synthetic biologists can produce a plethora of metabolites in cell factories in a jiff.

Self-decorating cells via surface-initiated enzymatic controlled radical polymerization.

Through the innovative use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of Saccharomyces cerevisiae cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant change in yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering.

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine

The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii

Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.

Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain

BackgroundLipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism.ResultsBuilding on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively.ConclusionsYields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

A designed bacterial microcompartment shell with tunable composition and precision cargo loading

Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as bacterial microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.

Diverse hydrocarbon biosynthetic enzymes can substitute for olefin synthase in the cyanobacterium Synechococcus sp. PCC 7002

Cyanobacteria are among only a few organisms that naturally synthesize long-chain alkane and alkene hydrocarbons. Cyanobacteria use one of two pathways to synthesize alka/enes, either acyl-ACP reductase (Aar) and aldehyde deformylating oxygenase (Ado) or olefin synthase (Ols). The genomes of cyanobacteria encode one of these pathways but never both, suggesting a mutual exclusivity. We studied hydrocarbon pathway compatibility using the model cyanobacterium Synechococcus sp. PCC 7002 (S7002) by co-expressing Ado/Aar and Ols and by entirely replacing Ols with three other types of hydrocarbon biosynthetic pathways. We find that Ado/Aar and Ols can co-exist and that slower growth occurs only when Ado/Aar are overexpressed at 38 °C. Furthermore, Ado/Aar and the non-cyanobacterial enzymes UndA and fatty acid photodecarboxylase are able to substitute for Ols in a knockout strain and conditionally rescue slow growth. Production of hydrocarbons by UndA in S7002 required a rational mutation to increase substrate range. Expression of the non-native enzymes in S7002 afforded unique hydrocarbon profiles and alka/enes not naturally produced by cyanobacteria. This suggests that the biosynthetic enzyme and the resulting types of hydrocarbons are not critical to supporting growth. Exchanging or mixing hydrocarbon pathways could enable production of novel types of CO2-derived hydrocarbons in cyanobacteria.

An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme

BackgroundStrategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism. Incorporating nonnative enzymes into host organisms can be challenging in cases such as this, where the enzyme has evolved in a very different environment from that of the host.ResultsIn this study, we use computational protein design to engineer the Zymomonas mobilis PDC to resist thermal denaturation at the growth temperature of a thermophilic host. We generate thirteen PDC variants using the Rosetta protein design software. We measure thermal stability of the wild-type PDC and PDC variants using circular dichroism. We then measure and compare enzyme endurance for wild-type PDC with the PDC variants at an elevated temperature of 60 °C (thermal endurance) using differential interference contrast imaging.ConclusionsWe find that increases in melting temperature (Tm) do not directly correlate with increases in thermal endurance at 60 °C. We also do not find evidence that any individual mutation or design approach is the major contributor to the most thermostable PDC variant. Rather, remarkable cooperativity among sixteen thermostabilizing mutations is key to rationally designing a PDC with significantly enhanced thermal endurance. These results suggest a generalizable iterative computational protein design approach to improve thermal stability and endurance of target enzymes.

A two-step evolutionary process establishes a non-native vitamin B6 pathway in Bacillus subtilis.

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.

An Enzyme‐Coated Metal–Organic Framework Shell for Synthetically Adaptive Cell Survival

A bioactive synthetic porous shell was engineered to enable cells to survive in an oligotrophic environment. Eukaryotic cells (yeast) were firstly coated with a β-galactosidase (β-gal), before crystallization of a metal-organic framework (MOF) film on the enzyme coating; thereby producing a bioactive porous synthetic shell. The β-gal was an essential component of the bioactive shell as it generated nutrients (that is, glucose and galactose) required for cell viability in nutrient-deficient media (lactose-based). Additionally, the porous MOF coating carried out other vital functions, such as 1) shielding the cells from cytotoxic compounds and radiation, 2) protecting the non-native enzymes (β-gal in this instance) from degradation and internalization, and 3) allowing for the diffusion of molecules essential for the survival of the cells. Indeed, this bioactive porous shell enabled the survival of cells in simulated extreme oligotrophic environments for more than 7 days, leading to a decrease in cell viability less than 30 %, versus a 99 % decrease for naked yeast. When returned to optimal growth conditions the bioactive porous exoskeleton could be removed and the cells regained full growth immediately. The construction of bioactive coatings represents a conceptually new and promising approach for the next-generation of cell-based research and application, and is an alternative to synthetic biology or genetic modification.

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins.

Plants, cyanobacteria, and algae generate a surplus of redox power through photosynthesis, which makes them attractive for biotechnological exploitations. While central metabolism consumes most of the energy, pathways introduced through metabolic engineering can also tap into this source of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic context.

Metabolic engineering of carbon and redox flow in the production of small organic acids.

The review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also depends on the feedstock used and cell energetic requirements for efficient metabolism during production. Recent work on the formation and commercial uses of a number of small mono- and diacids is discussed with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to improve yields. Since a number of widely studied acids derived from oxaloacetate as an important precursor, several of these acids are covered with the general strategies and particular components summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than succinate, the availability of reduction equivalents and level of aerobiosis are important parameters in optimizing production of these compounds in various hosts. Several other more oxidized acids are also discussed as in some cases, they may be desired products or their formation is minimized to afford higher yields of more reduced products. The placement and connections among acids in the typical central metabolic network are presented along with the use of a number of specific non-native enzymes to enhance routes to high production, where available alternative pathways and strategies are discussed. While many organic acids are derived from a few precursors within central metabolism, each organic acid has its own special requirements for high production and best compatibility with host physiology.

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.

GlycoSNAP: A High-Throughput Screening Methodology for Engineering Designer Glycosylation Enzymes.

The Campylobacter jejuni protein glycosylation locus (pgl) encodes enzymes for asparagine-linked (N-linked) glycosylation and serves as the prototype for N-glycosylation in bacteria. This pathway has been functionally transferred into Escherichia coli, thereby enabling efficient N-linked glycosylation of acceptor proteins with the C. jejuni heptasaccharide in this genetically tractable host. However, expansion of this bacterial system to encompass the vast biological diversity of potential glycans and target proteins often requires engineering of the non-native enzymes to isolate variants with optimal or altered functions. Here, we describe a genetic assay named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that facilitates engineering of glycosylation enzymes directly in E. coli. To date, this method has been successfully applied to screen (1) combinatorial libraries of bacterial oligosaccharyltransferases (OSTs) for relaxed substrate specificity and (2) acceptor site libraries to identify sequons recognized by natural OSTs as well as engineered variants.

Thermally induced inactivation and aggregation of urease: Experiments and population balance modelling

We present a population balance model for enzyme deactivation and aggregation kinetics with a limited number of physically relevant parameters and use this model to analyse the experimental data for thermal inactivation of jack bean urease. The time dependence of the relative enzymatic activity was found to follow the second order kinetics, which was consistent with pre-equilibrated folding/unfolding of the native enzyme, followed by irreversible cluster–cluster aggregation of the non-native enzyme resulting in gradual and permanent loss of enzymatic activity. Monomer–cluster aggregation scenario was considered but was not consistent with the observed kinetic order of monomer disappearance at longer times. We analysed time evolution of the average hydrodynamic radius obtained from dynamic light scattering measurements and by fitting these data with our model, we were able to estimate the value of the unfolding equilibrium constant with a reasonable accuracy ( K c ≈0.05 at 80 °C). We were also able to make order of magnitude estimates of the maximum number of enzyme molecules in the aggregated clusters (hundreds) as well as the aggregation rate constant of the non-native enzyme.

Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates

The health-beneficial effects of long-chain polyunsaturated fatty acids (LC-PUFAs), derived mainly from fish oil, coupled with the growing requirement for an alternative and sustainable source of these compounds, has led to efforts to engineer oilseed crops for their production. LC-PUFA synthesis has been achieved using combinations of heterologous endomembrane desaturases and elongases expressed in model oilseed plants. Two general approaches have been employed that both use endogenous 18 carbon fatty acids as the starting substrates: the Delta6- and Delta8-pathways, which perform desaturation followed by elongation or elongation followed by desaturation, respectively. However, yields above 20% have not yet been realized owing to bottlenecks that become apparent in the endogenous biosynthetic pathways when heterologous genes are expressed. These bottlenecks might be caused partly by inefficient non-native enzymes in the host system or also by suboptimal acyl-exchange mechanisms between the acyl-CoA and lipid class pools. The fine-tuning of the fatty acid flux between the acyl-CoA, phospholipid, and triacylglycerol pools will be essential to maximise polyunsaturated fatty acid yields in seed oils. In addition, efficient substrate channelling and lipid synthesis could depend on specific endoplasmic reticulum subdomain localisation for key endogenous enzymes, and this organization could be compromised in heterologous systems.

Influence of screening procedures on properties of monoclonal antibodies to glucose oxidase

Monoclonal antibodies to glucose oxidase (E.C. 1.1.3.4) from Aspergillus niger were prepared with apoenzyme as the antigen. Five of these antibodies, all of the IgG1 subisotype, were further characterized. They were affinity purified using an affinity column prepared by covalent immobilization of glucose oxidase via a spacer arm. Glucose oxidase lost its immunological reactivity with these monoclonal antibodies under direct covalent immobilization. The carbohydrate moiety of the enzyme is not immunogenic. None of the antibodies had any detectable effect on the catalytic properties of the enzyme. All the antibodies are directed towards segmental epitopes of the enzyme, and each enzyme subunit has more than one non-overlapping epitope. All five antibodies bound the non-native enzyme coated on ELISA plates in preference to the enzyme in solution.