ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA.

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

A wearable and flexible lactic-acid/O2 biofuel cell with an enhanced air-breathing biocathode.

The performance of biocathode in an enzymatic biofuel cell (EBFC) in the real application is somehow overlooked. Herein, a wearable and flexible lactic-acid/O2 EBFC enhanced with an air-breathing biocathode is designed to solve the limitation of biocathode that arises from the low solubility and slow mass transfer of the dissolved oxygen. To improve the oxygen supply efficiency for the air-breathing biocathode, a superhydrophobic base electrode creating an efficient air-solid-liquid triphase interface is developed. The designed EBFC with an 'island-bridge' configuration is integrated by assembling the current collectors of air-breathing biocathode and bioanode on a commercial laminating film (LF) screen-printed with a noninterfering circuit. It is found that the biocathode/bioanode area ratio should exceed 9:1 so that the designed EBFC (1A//9C) can achieve the optimal performance. This EBFC delivers an open circuit voltage of ca. 0.75 V and outputs a maximum power density of ca. 1.78 mW cm-2. In addition, a scaled-up EBFC (total bioanode area: 1.5 cm2) successfully powers a self-developed low-power device of heartrate in the pulse operation mode when applied on a volunteer's arm.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3.

Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Biosynthesis of artificial starch and microbial protein from agricultural residue.

Growing populations and climate change pose great challenges to food security. Humankind is confronting a serious question: how will we feed the world in the near future? This study presents an out-of-the-box solution involving the highly efficient biosynthesis of artificial starch and microbial proteins from available and abundant agricultural residue as new feed and food sources. A one-pot biotransformation using an in vitro coenzyme-free synthetic enzymatic pathway and baker's yeast can simultaneously convert dilute sulfuric acid-pretreated corn stover to artificial starch and microbial protein under aerobic conditions. The ß-glucosidase-free commercial cellulase mixture plus an ex vivo two-enzyme complex containing cellobiose phosphorylase and potato α-glucan phosphorylase displayed on the surface of Saccharomyces cerevisiae, showed better cellulose hydrolysis rates than a commercial ß-glucosidase-rich cellulase mixture. This is because the channeling of the hydrolytic product from the solid cellulosic feedstock to the yeast mitigated the inhibition of the cellulase cocktail. Animal tests have shown that the digestion of artificial amylose results in slow and relatively small changes in blood sugar levels, suggesting that it could be a new health food component that prevents obesity and diabetes. A combination of the utilization of available agricultural residue and the biosynthesis of starch and microbial protein from non-food biomass could address the looming food crisis in the food-energy-water nexus.

Carrier-Free Immobilization of Multi-Enzyme Complex Facilitates In†Vitro Synthetic Enzymatic Biosystem for Biomanufacturing.

A method is developed for carrier-free immobilization of multi-enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher-SpyTag and dockerin-cohesin) and enzyme component self-oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher-SpyTag and cohesins are prepared to recruit the four dockerin-containing cascade enzymes (i. e., alpha-glucan phosphorylase, phosphoglucomutase, inositol 1-phosphate synthase, and inositol 1-phosphatase) that can convert starch into inositol, forming multi-enzyme complexes. These self-assembled enzyme complexes show higher initial reaction rates than the four-enzyme cocktail. Moreover, water-insoluble self-assembled multi-enzyme complexes are observed, being the carrier-free immobilized multi-enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier-free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.

Engineering of a thermophilic dihydroxy-acid dehydratase toward glycerate dehydration for in vitro biosystems.

Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. KEY POINTS: • A screening method was established for the protein engineering of SsDHAD. • A R3 mutant of SsDHAD with 10-fold higher activity was obtained. • The R3 mutant exhibits higher productivity in the in vitro biosystems.

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis.

To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25-40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.

Enzymatic regeneration and conservation of ATP: challenges and opportunities.

Adenosine triphosphate (ATP), the universal energy currency of life, has a central role in numerous biochemical reactions with potential for the synthesis of numerous high-value products. ATP can be regenerated by three types of mechanisms: substrate level phosphorylation, oxidative phosphorylation, and photophosphorylation. Current ATP regeneration methods are mainly based on substrate level phosphorylation catalyzed by one enzyme, several cascade enzymes, or in vitro synthetic enzymatic pathways. Among them, polyphosphate kinases and acetate kinase, along with their respective phosphate donors, are the most popular approaches for in vitro ATP regeneration. For in vitro artificial pathways, either ATP-free or ATP-balancing strategies can be implemented via smart pathway design by choosing ATP-independent enzymes. Also, we discuss some remaining challenges and suggest perspectives, especially for industrial biomanufacturing. Development of ATP regeneration systems featuring low cost, high volumetric productivity, long lifetime, flexible compatibility, and great robustness could be one of the bottom-up strategies for cascade biocatalysis and in vitro synthetic biology.

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme.

Hyperthermophilic archaea with unique biochemical and physiological characteristics are important organisms for fundamental research of life science and have great potential for biotechnological applications. However, low transformation efficiency of foreign DNA molecules impedes developments in genetic modification tools and industrial applications. In this study, we applied prolonged overlap extension PCR (POE-PCR) to generate multimeric DNA molecules and then transformed them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1. This study was the first example to demonstrate the enhanced transformation efficiencies of POE-PCR products by a factor of approximately 100 for T. kodakarensis KOD1 and 8 for P. yayanosii A1, respectively, relative to circular shuttle plasmids. Furthermore, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), was conducted to obtain more stable ones due to high transformation efficiency of T. kodakarensis (i.e. ~3 × 104  CFU per µg DNA). T. kodakarensis harbouring the most thermostable MoHMGR mutant can grow in the presence of a thermostable antibiotic simvastatin at 85°C and even higher temperatures. This high transformation efficiency technique could not only help develop more hyperthermophilic enzyme mutants via directed evolution but also simplify genetical modification of archaea, which could be novel hosts for industrial biotechnology.

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

CO2 fixation for malate synthesis energized by starch via in vitro metabolic engineering.

Carbon dioxide (CO2) is an appealing carbon feedstock for the sustainable production of biocommodities. Here we designed three in vitro artificial enzymatic pathways featuring the ATP-excess, ATP-deficit, and ATP-balanced pathways for the biotransformation of starch and CO2 to malate. This ATP-balanced pathway without exogenous ATP donors can auto-regulate its carbon fluxes from glyceraldehyde 3-phosphate to 3-phosphoglycerate via either the ATP-generating pathway (a part of glycolysis) or no-ATP-generating pathway from a hyperthermophilic archaeon Thermococcus kodakarensis. The ATP-balanced pathway enabled to produce up to 52.4 mM malate with 95.3% of the theoretical yield, that is, 2 mol of malate synthesized from 1 mol of glucose of starch and 2 mol of CO2. This pathway also enabled to produce high-yield malate regardless of ATP/ADP ratios. Anaerobic reaction conditions and/or the addition of a reducing agent dithiothreitol were of importance for creating an anoxic environment for biocatalysis of enzyme cocktails and for mitigating the deactivation of enzymes and degradation of intermediates. This new pathway could provide a green route for direct conversion of CO2 to many building blocks, a promising alternative of petrochemical-based production of biocommodities.

A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2 ). Here, the authors expressed the four αßγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

Upgrade of wood sugar d-xylose to a value-added nutraceutical by in vitro metabolic engineering.

The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.

Construction of Enzyme-Cofactor/Mediator Conjugates for Enhanced in Vitro Bioelectricity Generation.

Cofactor-dependent oxidoreduction and electron transfer play an important role in in vitro bioelectricity generation and many other enzyme biocatalysis reactions. To facilitate such electron generation and transfer, several approaches based on the coimmobilization of cofactors and oxidoreductases have been demonstrated. Herein, a convenient and immobilization-free approach of constructing enzyme-cofactor and enzyme-mediator conjugates was developed. The in vitro bioelectricity generation reactions via enzymatic fuel cells were evaluated. The cells equipped by the conjugates exhibited significantly improved power output and stability in contrast to those mediated by unconjugated enzymes. These results may bring a new avenue in constructing efficient in vitro electron transfer chains for various biocatalysis applications.

An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design.

Although most in vitro (cell-free) synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products, in vitro synthetic biology platform, which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes, has been proposed for low-cost biomanufacturing of bioenergy, food, biochemicals, and nutraceuticals. In addition to the most important advantage-high product yield, in vitro synthetic biology platform features several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this article, we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks (thermoenzymes and/or immobilized enzymes) to building modules (e.g., enzyme complexes or multiple enzymes as a module) with specific functions. With development in thermostable building blocks-BioBricks and modules, the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.

Conversion of d-glucose to l-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions.

Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates. Here, a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of d-glucose to l-lactate via pyruvate. This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation. After optimization of enzyme loading amounts, buffer concentration and cofactor concentration, d-glucose was converted to l-lactate with a product yield of ∼90%. Our study has provided an emerging platform with potentials in producing pyruvate-derived chemicals, and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In†Vitro Hydrogen Production at Elevated Temperature.

To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX.

Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.