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.

Arginine Is a Novel Drug Target for Arginine Decarboxylase in Human Colorectal Cancer Cells.

Colorectal cancer (CRC) has been proven to be highly reliant on arginine availability. Limiting arginine-rich foods or treating patients with arginine-depleting enzymes arginine deiminase (ADI) or arginase can suppress colon cancer. However, arginase and ADI are not the best drug candidates for CRC. Ornithine, the product of arginase, can enhance the supply of polyamine, which favors CRC cell growth, while citrulline, the product of ADI, faces the problem of arginine recycling due to the overexpression of argininosuccinate synthetase (ASS). Biosynthetic arginine decarboxylase (ADC), an enzyme that catalyzes the conversion of arginine to agmatine and carbon dioxide, may be a better choice as it combines both arginine depletion and suppression of intracellular polyamine synthesis via its product agmatine. ADC has anti-tumor potential yet has received much less attention than the other two arginine-depleting enzymes. In order to gain a better understanding of ADC, the preparation and the anti-cancer properties of this enzyme were explored in this study. When tested in vitro, ADC inhibited the proliferation of three colorectal cancer cell lines regardless of their ASS cellular expression. In contrast, ADC had a lesser cytotoxic effect on the human foreskin fibroblasts and rat primary hepatocytes. Further in vitro studies revealed that ADC induced S and G2/M phase cell-cycle arrest and apoptosis in HCT116 and LoVo cells. ADC-induced apoptosis in HCT116 cells followed the mitochondrial apoptotic pathway and was caspase-3-dependent. With all results obtained, we suggest that arginine is a potential target for treating colorectal cancer with ADC, and the anti-cancer properties of ADC should be more deeply investigated in the future.

Biomanufacturing by In Vitro Biotransformation (ivBT) Using Purified Cascade Multi-enzymes.

In vitro biotransformation (ivBT) refers to the use of an artificial biological reaction system that employs purified enzymes for the one-pot conversion of low-cost materials into biocommodities such as ethanol, organic acids, and amino acids. Unshackled from cell growth and metabolism, ivBT exhibits distinct advantages compared with metabolic engineering, including but not limited to high engineering flexibility, ease of operation, fast reaction rate, high product yields, and good scalability. These characteristics position ivBT as a promising next-generation biomanufacturing platform. Nevertheless, challenges persist in the enhancement of bulk enzyme preparation methods, the acquisition of enzymes with superior catalytic properties, and the development of sophisticated approaches for pathway design and system optimization. In alignment with the workflow of ivBT development, this chapter presents a systematic introduction to pathway design, enzyme mining and engineering, system construction, and system optimization. The chapter also proffers perspectives on ivBT development.

A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis.

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.

[Advances in a new energy system based on electricity-hydrogen-carbohydrate cycle].

The development of green and low-carbon renewable energy systems has become an important international consensus. It is also an essential path for China to implement the dual-carbon strategy, ensure national energy security, and achieve sustainable development. This review introduces the theory of a new energy system based on electricity-hydrogen-carbohydrate (EHC) cycle, and highlights the biotransformations of carbohydrate/water-to-hydrogen, carbohydrate-to-electricity, and CO2-to-carbohydrate powered by hydrogen- or electric-energy based on the in vitro synthetic enzymatic biosystems (ivSEB) developed by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade. We elaborate the design principle and the molecular basis of ivSEB, and further expand from the EHC cycle to in vitro biomanufacturing with starch as the feedstock. Combined with the latest research advances, we analyze and discuss advantages and disadvantages of ivSEB, prospect future directions, so as to promote the green, low-carbon and sustainable development of economy and society.

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems.

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields ß-glucose 1-phosphate (ß-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitro synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymatic reaction module comprised of MP, ß-phosphoglucomutase (ß-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm2, whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.

Installing a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) Synthesis.

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co-substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light-energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.

Cell-free chemoenzymatic starch synthesis from carbon dioxide.

Starches, a storage form of carbohydrates, are a major source of calories in the human diet and a primary feedstock for bioindustry. We report a chemical-biochemical hybrid pathway for starch synthesis from carbon dioxide (CO2) and hydrogen in a cell-free system. The artificial starch anabolic pathway (ASAP), consisting of 11 core reactions, was drafted by computational pathway design, established through modular assembly and substitution, and optimized by protein engineering of three bottleneck-associated enzymes. In a chemoenzymatic system with spatial and temporal segregation, ASAP, driven by hydrogen, converts CO2 to starch at a rate of 22 nanomoles of CO2 per minute per milligram of total catalyst, an ~8.5-fold higher rate than starch synthesis in maize. This approach opens the way toward future chemo-biohybrid starch synthesis from CO2.

An ATP-free in vitro synthetic enzymatic biosystem facilitating one-pot stoichiometric conversion of starch to mannitol.

D-Mannitol (hereinafter as mannitol) is a six-carbon sugar alcohol with diverse applications in food and pharmaceutical industries. To overcome the drawbacks of the chemical hydrogenation method commonly used for mannitol production at present, there is a need to search for novel prospective mannitol production strategies that are of high yield and low cost. In this study, we present a novel approach for the stoichiometric synthesis of mannitol via an in vitro synthetic enzymatic biosystem using the low-cost starch as substrate. By dividing the overall reaction pathway into three modules which could be executed sequentially in one pot, our design aimed at the stoichiometric conversion of starch-based materials into mannitol in an ATP-independent and cofactor-balanced manner. At optimized conditions, high product yields of around 95-98% were achieved using both 10 g/L and 50 g/L maltodextrin as substrate, indicating the potential of our designed system for industrial applications. This study not only provides a high-efficient strategy for the synthesis of mannitol but also expands the product scope of sugar alcohols by the in vitro synthetic enzymatic biosystems using low-cost starch-based materials as the input. KEY POINTS : • We described a design-build-test-learn pipeline to construct in vitro biosystems. • The designed system comprised six key enzymes and another three enzymes. • The system converted maltodextrin stoichiometrically to mannitol in one pot.

Construction of an Artificial In Vitro Synthetic Enzymatic Platform for Upgrading Low-Cost Starch to Value-Added Disaccharides.

Disaccharides are valuable oligosaccharides with an increasing demand in the food, cosmetic, and pharmaceutical industries. Disaccharides can be manufactured by extraction from the acid hydrolysate of plant-derived substrates, but this method has several issues, such as the difficulty in accessing natural substrates, laborious product separation processes, and troublesome wastewater treatment. A chemical synthesis using glucose was developed for producing disaccharides, but this approach suffers from a low product yield due to the low specificity and requires tedious protection and deprotection processes. In this study, we adopted an artificial strategy for producing a variety of value-added disaccharides from low-cost starch through the construction of an in vitro synthetic enzymatic platform: two enzymes worked in parallel to convert starch to glucose and glucose 1-phosphate, and these two intermediates were subsequently condensed together to a disaccharide by a disaccharide phosphorylase. Several disaccharides, such as laminaribiose, cellobiose, trehalose, and sophorose, were produced successfully from starch with the yields of more than 80% with the help of kinetic mathematical models to predict the optimal reaction conditions, exhibiting great potential in an industrial scale. This study provided a promising alternative to reform the mode of disaccharide manufacturing.

[In vitro multi-enzyme molecular machines - a review].

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.

Facile synthesis of (-)-vibo-quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem.

(-)-vibo-Quercitol (VQ: 1L-1,2,4/3,5-cyclohexanepentol), a form of deoxyinositol, is an alternative chiral building block in the synthesis of bioactive compounds to control diabetes. In this study, an adenosine triphosphate-free in vitro synthetic enzymatic biosystem composed of five enzymes (including one enzyme for NADH regeneration) was constructed to produce VQ from maltodextrin in one-pot. After optimization of reaction conditions, 7.6 g/L VQ was produced from 10 g/L maltodextrin with a product yield (mol/mol) of 77%, and 25.3 g/L VQ with a purity of 87% was produced from 50 g/L maltodextrin through simple scaling up of this nonfermentative enzymatic biosystem. Therefore, this study provides an economical and environmentally friendly method for the envisioned quercitol biosynthesis.

Enzymatic characterization of a thermostable phosphatase from Thermomicrobium roseum and its application for biosynthesis of fructose from maltodextrin.

Phosphatases, which catalyze the dephosphorylation of compounds containing phosphate groups, are important members of the haloacid dehalogenase (HAD)-like superfamily. Herein, a thermostable phosphatase encoded by an open reading frame of Trd_1070 from Thermomicrobium roseum was enzymologically characterized. This phosphatase showed promiscuous activity against more than ten sugar phosphates, with high specific activity toward ribose 5-phosphate, followed by ribulose 5-phosphate and fructose 6-phosphate. The half-life of Trd_1070 at 70 °C and pH 7.0 was about 14.2 h. Given that the catalytic efficiency of Trd_1070 on fructose 6-phosphate was 49-fold higher than that on glucose 6-phosphate, an in vitro synthetic biosystem containing alpha-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, and Trd_1070 was constructed for the production of fructose from maltodextrin by whole-cell catalysis, resulting in 21.6 g/L fructose with a ratio of fructose to glucose of approximately 2:1 from 50 g/L maltodextrin. This in vitro biosystem provides an alternative method to produce fructose with higher fructose content compared with the traditional production method using glucose isomerization. Further discovery and enzymologic characterization of phosphatases may promote further production of alternative monosaccharides through in vitro synthetic biosystems.

The Construction of an In Vitro Synthetic Enzymatic Biosystem that Facilitates Laminaribiose Biosynthesis from Maltodextrin and Glucose.

Laminaribiose is a reducing disaccharide linked by a ß-1,3 glycosidic bond; it is also a precursor for building blocks in the pharmaceutical industry, a powerful germinating agent and antiseptic, as well as a potential prebiotic. In this study, an in vitro enzymatic biosystem composed of α-glucan phosphorylase, laminaribiose phosphorylase, isoamylase, and 4-glucanotransferase is designed for the one-pot synthesis of laminaribiose from low-cost maltodextrin and glucose. Through condition optimization, 51 mM laminaribiose is produced from 10 g L-1 maltodextrin (55.5 mM glucose equivalent) and 90 mM glucose. The product yield based on maltodextrin is 91.9%. To investigate the industrial potential of this in vitro enzymatic biosystem, the production of laminaribiose from high concentrations of substrates is also examined, and 179 mM laminaribiose is produced from 50 g L-1 of maltodextrin and 450 mM glucose. This in vitro enzymatic biosystem comprised of thermophilic enzymes can drastically decrease the manufacturing cost of laminaribiose and provide a green method for the production of other disaccharides using phosphorylases.

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.

Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology.

Two natural nicotinamide-based coenzymes (NAD and NADP) are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism, respectively. Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles. This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways. Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering, which is closely related to product-oriented synthetic biology projects. This review focuses on the methodology of nicotinamide-based coenzyme engineering, with its application in improving product yields and decreasing production costs. Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes. Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review. Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering, especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.