Complete genome sequence of Paraburkholderia terrae strain KU-46, a 2,4-dinitrophenol-degrading bacterium.

Paraburkholderia terrae strain KU-46 has been studied for its capability to degrade 2,4-dinitrophenol. Here, we present the complete 10,833,180bp genome of this microorganism, comprising five circular chromosomes housing 9,797 protein-coding sequences. The genes responsible for 2,4-dinitrophenol and 4-nitrophenol degradation are located on chromosome 2.

Precise microbiome engineering using natural and synthetic bacteriophages targeting an artificial bacterial consortium.

In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum, the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 102 orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the λ phage was synthesized from prophage DNA in the genome of λ lysogen by in vivo DNA assembly and phage-rebooting techniques, and E. coli-targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples.

Effects of small heat shock proteins from thermotolerant bacteria on the stress resistance of Escherichia coli to temperature, pH, and hyperosmolarity.

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

Identification and characterization of a pab gene cluster responsible for the 4-aminobenzoate degradation pathway, including its involvement in the formation of a γ-glutamylated intermediate in Paraburkholderia terrae strain KU-15.

Paraburkholderia terrae strain KU-15 grows on 2- and 4-nitrobenzoate and 2- and 4-aminobenzoate (ABA) as the sole nitrogen and carbon sources. The genes responsible for the potential degradation of 2- and 4-nitrobenzoate and 2-ABA have been predicted from its genome sequence. In this study, we identified the pab operon in P. terrae strain KU-15. This operon is responsible for the 4-ABA degradation pathway, which involves the formation of a γ-glutamylated intermediate. Reverse transcription-polymerase chain reaction revealed that the pab operon was induced by 4-ABA. Herein, studying the deletion of pabA and pabB1 in strain KU-15 and the examining of Escherichia coli expressing the pab operon revealed the involvement of the operon in 4-ABA degradation. The first step of the degradation pathway is the formation of a γ-glutamylated intermediate, whereby 4-ABA is converted to γ-glutamyl-4-carboxyanilide (γ-GCA). Subsequently, γ-GCA is oxidized to protocatechuate. Overexpression of various genes in E. coli and purification of recombinant proteins permitted the functional characterization of relevant pathway proteins: PabA is a γ-GCA synthetase, PabB1-B3 functions in a multicomponent dioxygenase system responsible for γ-GCA dioxygenation, and PabC is a γ-GCA hydrolase that reverses the formation of γ-GCA by PabA.

Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

Subtractive modification of bacterial consortium using antisense peptide nucleic acids.

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

Dietary-protein sources modulate host susceptibility to Clostridioides difficile infection through the gut microbiota.

Clostridioides difficile causes nosocomial antibiotic-associated diarrhea on a global scale. Susceptibility to C. difficile infection (CDI) is influenced by the composition and metabolism of gut microbiota, which in turn are affected by diet. However, the mechanism underlying the interplay between diet and gut microbiota that modulates susceptibility to CDI remains unclear. Here, we show that a soy protein diet increases the mortality of antibiotic-treated, C. difficile-infected mice while also enhancing the intestinal levels of amino acids (aas) and relative abundance of Lactobacillus genus. Indeed, Ligilactobacillus murinus-mediated fermentation of soy protein results in the generation of aas, thereby promoting C. difficile growth, and the process involves the anchored cell wall proteinase PrtP. Thus, mutual interaction between dietary protein and the gut microbiota is a critical factor affecting host susceptibility to CDI, suggesting that dietary protein sources can be an important determinant in controlling the disease.

One-Step Preparation of Cell-Free ATP Regeneration Module Based on Non-Oxidative Glycolysis Using Thermophilic Enzymes.

Adenosine triphosphate (ATP) is an essential cofactor for energy-dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non-oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597-5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co-expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one-step purification via a heat-treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co-expression vectors.

Complete Genome Sequence of Paraburkholderia terrae Strain KU-15, a 2-Nitrobenzoate-Degrading Bacterium.

Paraburkholderia terrae strain KU-15 has been investigated for its ability to degrade 2-nitrobenzoate. Here, we report the complete 10,422,345-bp genome of this microorganism, which consists of six circular replicons containing 9,483 protein-coding sequences. The genome carries genes that are potentially responsible for 2-nitrobenzoate and 4-nitirobenzoate degradation.

l-Lactate oxidase-mediated removal of l-lactic acid derived from fermentation medium for the production of optically pure D-lactic acid.

BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.

Improvement of production yield of l-cysteine through in vitro metabolic pathway with thermophilic enzymes.

The demand for the amino acid l-cysteine is increasing in the food, cosmetic, and pharmaceutical industries. Conventionally, the commercial production of l-cysteine is achieved by its extraction from the acid hydrolysate of hair and feathers. However, this production method is associated with the release of environmentally hazardous wastewater. Additionally, l-cysteine produced from animal sources cannot be halal-certified, which limits the market size. Although recent studies have developed an alternative commercial l-cysteine production method based on microbial fermentation, the production yield was insufficient owing to the cytotoxicity of l-cysteine against the host cells. In a previous study, we had developed an in vitrol-cysteine production method with a combination of 11 thermophilic enzymes, which yielded 10.5 mM l-cysteine from 20 mM glucose. In this study, we performed re-screening for enzymes catalyzing the rate-limiting steps of the in vitro pathway. Subsequently, the genes encoding enzymes necessary for the in vitro synthesis of l-cysteine were assembled in an expression vector and co-expressed in a single strain. To prevent the synthesis of hydrogen peroxide (H2O2), which is a byproduct and inhibits the enzyme activity, the redox balance in this biosynthetic pathway was maintained by replacing the H2O2-forming NADH oxidase with another enzymatic reaction in which pyruvate was used as a sacrificial substrate. The re-designed in vitro synthetic pathway resulted in the production of 28.2 mM l-cysteine from 20 mM glucose with a molar yield of 70.5%.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli.

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA A-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaB A-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaC A-04, 1770 bp), was identified. Sequence analysis of the phaA A-04, phaB A-04, and phaCA-04 genes revealed that phaC A-04 was 99% similar to phaC H16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaC A-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCAB A-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCAB A-04 and pColdTF-phaCAB A-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y P/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCAB A-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y P/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCAB A-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Genome editing by miniature CRISPR/Cas12f1 enzyme in Escherichia coli.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.

In Vitro Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.

TEMPURA: Database of Growth TEMPeratures of Usual and RAre Prokaryotes.

Growth temperature is one of the most representative biological parameters for characterizing living organisms. Prokaryotes have been isolated from various temperature environments and show wide diversity in their growth temperatures. We herein constructed a database of growth TEMPeratures of Usual and RAre prokaryotes (TEMPURA, http://togodb.org/db/tempura), which contains the minimum, optimum, and maximum growth temperatures of 8,639 prokaryotic strains. Growth temperature information is linked with taxonomy IDs, phylogenies, and genomic information. TEMPURA provides useful information to researchers working on biotechnological applications of extremophiles and their biomolecules as well as those performing fundamental studies on the physiological diversity of prokaryotes.

In vitro reconstitution of non-phosphorylative Entner-Doudoroff pathway for lactate production.

In vitro metabolic engineering is an emerging framework for bioproduction systems, in which synthetic metabolic pathways are constructed using a limited number of enzymes. Employment of thermophilic enzymes as catalytic elements in pathways enables the use of simple heat purification of recombinantly expressed enzymes. However, thermophilic enzymes are generally incompatible with thermo-labile substrates and intermediates. In previous work, we showed that lactate production through a non-ATP forming chimeric Embden-Meyerhof (EM) pathway required careful adjustment of the metabolic fluxes by continuous substrate feeding and optimization of enzyme ratios to prevent the accumulation and degradation of thermo-labile intermediates (Ye et al., Microb. Cell Fact., 11, 120, 2012). In the study reported here, we constructed an in vitro non-phosphorylative Entner-Doudoroff (np-ED) pathway. Because of the high thermal stability of the metabolic intermediates in the np-ED pathway, it could prevent degradation of accumulated metabolic intermediates caused by inconstant metabolic fluxes, and batch-mode production of lactate in which the concentrations of the substrate and metabolic intermediates change dynamically could be achieved. By combining the enzymes involved in the np-ED pathway and lactate dehydrogenase, 20.9 mM lactate was produced from 10 mM glucose and 1 mM gluconate in 6 h.

In vitro production of cysteine from glucose.

Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.

Developing a single strain for in vitro salvage synthesis of NAD+ at high temperatures and its potential for bioconversion.

BACKGROUND: Thermostable enzymes have several advantages over their mesophilic counterparts for industrial applications. However, trade-offs such as thermal instability of enzyme substrates or co-factors exist. Nicotinamide adenine dinucleotide (NAD+) is an important co-factor in many enzyme-catalyzed oxidation-reduction reactions. This compound spontaneously decomposes at elevated temperatures and basic pH, a property that limits catalysis of NAD+/NADH-dependent bioconversions using thermostable enzymes to short timeframes. To address this issue, an "in vitro metabolic pathway" for salvage synthesis of NAD+ using six thermophilic enzymes was constructed to resynthesize NAD+ from its thermal decomposition products at high temperatures. RESULTS: An integrated strain, E. coli DH5α (pBR-CI857, pGETS118-NAD+), that codes for six thermophilic enzymes in a single operon was constructed. Gene-expression levels of these enzymes in the strain were modulated by their sequential order in the operon. An enzyme solution containing these enzymes was prepared by the heat purification from the cell lysate of the integrated strain, and used as an enzyme cocktail for salvage synthesis of NAD+. The salvage activity for synthesis of NAD+ from its thermal decomposition products was found to be 0.137 ± 0.006 µmol min-1 g-1 wet cells. More than 50% of this initial activity remained after 24 h at 60 °C. The enzyme cocktail could maintain a NAD+ concentration of 1 mM for 12 h at 60 °C. Furthermore, this enzyme cocktail supported continuous NAD+/NADH-dependent redox reactions using only NAD+/NADH derived from host cells, without the need for addition of external NAD+. CONCLUSIONS: The integrated strain allows preparation of an enzyme cocktail that can solve the problem of NAD+ instability at high temperatures. The strain simplifies preparation of the enzyme cocktail, and thus expands the applicability of the in vitro metabolic engineering method using thermostable enzymes. Further optimization of gene expressions in the integrated strain can be achieved by using various types of ribosome binding sites as well as promoters.