Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli.

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD+ also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD+-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD+-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD+ by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD+ synthesis were overexpressed and led to a 221% increase in the NAD+ concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD+ content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD+ by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD+ was constructed, with the intracellular NAD+ concentration reaching 26.9 µmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD+-producing strain through global regulation metabolic engineering.

Enhanced catalytic efficiency and coenzyme affinity of leucine dehydrogenase by comprehensive screening strategy for L-tert-leucine synthesis.

L-tert-leucine (L-Tle) is widely used as vital chiral intermediate for pharmaceuticals and as chiral auxiliarie for organocatalysis. L-Tle is generally prepared via the asymmetric reduction of trimethylpyruvate (TMP) catalyzed by NAD+-dependent leucine dehydrogenase (LeuDH). To improve the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus, mutation libraries constructed by error-prone PCR and iterative saturation mutation were screened by two kinds of high-throughput methods. Compared with the wild type, the affinity of the selected mutant E24V/E116V for TMP and NADH increased by 7.7- and 2.8-fold, respectively. And the kcat/Km of E24V/E116V on TMP was 5.4-fold higher than that of the wild type. A coupled reaction comprising LeuDH with glucose dehydrogenase of Bacillus amyloliquefaciens resulted in substrate inhibition at high TMP concentrations (0.5 M), which was overcome by batch-feeding of the TMP substrate. The total turnover number and specific space-time conversion of 0.57 M substrate increased to 11,400 and 22.8 mmol·h-1·L-1·g-1, respectively. KEY POINTS: • The constructed new high-throughput screening strategy takes into account the two indicators of catalytic efficiency and coenzyme affinity. • A more efficient leucine dehydrogenase (LeuDH) mutant (E24V/E116V) was identified. • E24V/E116V has potential for the industrial synthesis of L-tert-leucine.

Optimization and Validation of a Headspace Solid-Phase Microextraction with Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometric Detection for Quantification of Trace Aroma Compounds in Chinese Liquor (Baijiu).

The detection of trace aroma compounds in samples with complex matrices such as Chinese liquor (Baijiu) requires a combination of several methods, which makes the analysis process very complicated. Therefore, a headspace solid-phase microextraction (HS-SPME) method coupled with two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) was developed for the quantitation of a large number of trace compounds in Baijiu. Optimization of extraction conditions via a series of experiments revealed that dilution of the alcohol content of 8 mL of Baijiu to 5%, followed by the addition of 3.0 g of NaCl and subsequent SPME extraction with DVB/CAR/PDMS fiber coating over 45 min at 45 °C was the most suitable. To check the matrix effects, various model Baijiu matrices were investigated in detail. The quantitative method was established through an optimized model synthetic solution, which can identify 119 aroma compounds (esters, alcohols, fatty acids, aldehydes and ketones, furans, pyrazines, sulfur compounds, phenols, terpenes, and lactones) in the Baijiu sample. The developed procedure provided high recovery (86.79-117.94%), good repeatability (relative standard deviation < 9.93%), high linearity (R2 > 0.99), and lower detection limits than reported methods. The method was successfully applied to study the composition of volatile compounds in different types of Baijiu. This research indicated that the optimized HS-SPME-GC×GC-TOFMS method was a valid and accurate procedure for the simultaneous determination of different types of trace compounds in Baijiu. This developed method will allow an improved analysis of other samples with complex matrices.

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids.

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products.

[High efficient co-expression of leucine dehydrogenase and glucose dehydrogenase in Escherichia coli].

Objective: Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine. Methods: The leucine dehydrogenase (ldh) from Bacillus cereus and glucose dehydrogenase (gdh) from Bacillus sp. were co-expressed by three different strategies, including co-expressing two genes in single vector, co-expressing two genes in two vectors and expressing fusion protein. The catalytic efficiencies of recombinant strains with different enzyme activity ratio in different modes of biocatalyst were compared to produce L-tert-leucine from its corresponding α-keto acids. Results: Different co-expression strategies displayed a slight impact on leucine dehydrogenase expression, whereas, a greater impact on glucose dehydrogenase. All the activity of leucine dehydrogenase was normally expressed, but the fusion proteins lost the activity of glucose dehydrogenase. Besides, the activity of glucose dehydrogenase was also totally inhibited when the 6-histidine tag was fused at C termini, which indicated the additional 6-histidine tag considerately depressed the glucose dehydrogenase activity. After optimization of expression, three recombinant strains exhibiting high enzyme activity and different enzyme activity ratio were used to synthesis L-tert-leucine in the mode of cell-free extracts and whole-cell. Result displayed a great influence on the catalytic efficiencies resulted from the mode of catalyst and enzyme activity. When the cell-free crude culture broth of E. coli BL21/pET28a-L-SD-AS-G coexpressing two genes in single vector was used as biocatalyst, 15 g/L cell loading and 0.1 mmol/L NAD+ were enough to completely transform 0.5 mol/L trimethylpyruvate into L-tert-leucine. Conclusion: The recombinant strain with high activities of leucine dehydrogenase and glucose dehydrogenase was achieved by co-expressing two genes in single vector without histidine tag in E. coli and L-tert-leucine was efficiently produced with this recombinant strain.

Breeding of a cyclic imide-assimilating bacterium, Pseudomonas putida s52, for high efficiency production of pyruvate.

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.

High coenzyme affinity chimeric amine dehydrogenase based on domain engineering.

NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from phenylalanine dehydrogenase (PheDH) catalyzes the synthesis of aromatic chiral amines from prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit its industrial application. Here, we developed a chimeric amine dehydrogenase, cFLF-AmDH, based on the relative independence of the structure at the domain level, combined with a substrate-binding domain from F-AmDH and a high-affinity cofactor-binding domain from leucine amine dehydrogenase (L-AmDH). The kinetic parameters indicated that cFLF-AmDH showed a twofold improvement in affinity for NADH and a 4.4-fold increase in catalytic efficiency (kcat/Km) compared with the parent F-AmDH. Meanwhile, cFLF-AmDH also showed higher thermal stability, with the half-life increased by 60% at 55 °C and a broader substrate spectrum, than the parent F-AmDH. Molecular dynamics simulations suggested that the constructed cFLF-AmDH had a more stable structure than the parent F-AmDH, thereby improving the affinity of the coenzyme. The reaction rate increased by 150% in the reductive amination reaction catalyzed by cFLF-AmDH. When the NAD+ concentration was 0.05 mM, the conversion rate was increased by 150%. These results suggest that the chimeric protein by domain shuffling from different domain donors not only increased the cofactor affinity and catalytic efficiency, but also changed the specificity and thermal stability. Our study highlights that domain engineering is another effective method for creating biodiversity with different catalytic properties.

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase.

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the k cat/K m of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

BACKGROUND: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. RESULTS: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmol g-1 L-1 h-1. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. CONCLUSIONS: Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

Integration of newly isolated biocatalyst and resin-based in situ product removal technique for the asymmetric synthesis of (R)-methyl mandelate.

The enantioselective reduction of methyl benzoylformate to (R)-methyl mandelate, an important pharmaceutical intermediate and a versatile resolving agent, was investigated in this study. After minimizing the reaction-specific constraints (constraints dependent on the nature of the substrate and product) by preliminary selection of the reaction parameters, an effective whole cell biocatalyst (Saccharomyces cerevisiae AS2.1392) was obtained by simple screening procedures. Under further optimized conditions, a product concentration of 103 mmol L(-1) could be attained within 5 h with a yield of 85.8% and an enantiometric excess of 95.4%, indicating S. cerevisiae AS2.1392 an efficient biocatalyst for the asymmetric synthesis of (R)-methyl mandelate. Furthermore, resin-based in situ product removal (ISPR) technique was applied to alleviate the substrate and product inhibition or toxicity to the whole cells. The integration of newly isolated biocatalyst and proper ISPR technique provides a practical route for the preparation of optically active pharmaceutical intermediates.

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system.

An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.e. and yield of 90% after 60 h. Simultaneously, it was observed that change in cell morphology impedes the further enhancement of substrate concentration in this system but can be reversibly changed after stereoinversion or cultivation in systems without PEG.

Crystallization and preliminary X-ray crystallographic analysis of a carbonyl reductase from Candida parapsilosis.

A novel short-chain NADPH-dependent (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) has been crystallized. Two distinct but related crystal forms of SCR were obtained using the hanging-drop vapour-diffusion method and a reservoir solution consisting of 18%(w/v) polyethylene glycol 2000 monomethyl ether and 8%(v/v) 2-propanol as the precipitant. The crystals were rhomboid in shape with average dimensions of 0.3 x 0.3 x 0.4 mm and diffracted to a resolution of 2.7-3.0 A. The crystal forms both belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 104.7, b = 142.8, c = 151.8 A and a = 101.1, b = 146.0, c = 159.8 A. The calculated values of V(M), rotation-function and translation-function solutions and consideration of potential crystal packing suggest that there are eight protein subunits per asymmetric unit.

Disorder prediction-based construct optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase.

Pullulanase is a well-known starch-debranching enzyme. However, the production level of pullulanase is yet low in both wide-type strains and heterologous expression systems. We predicted the disorder propensities of Bacillus naganoensis pullulanase (PUL) using the bioinformatics tool, Disorder Prediction Meta-Server. On the basis of disorder prediction, eight constructs, including PULΔN5, PULΔN22, PULΔN45, PULΔN64, PULΔN78 and PULΔN106 by deleting the first 5, 22, 45, 64, 78 and 106 residues from the N-terminus, and PULΔC9 and PULΔC36 by deleting the last 9 and 36 residues from the C-terminus, were cloned into the recombinant expression vector pET-28a-PelB and auto-induced in Escherichia coli BL21 (DE3) cells. All constructs were evaluated in production level, specific activities and kinetic parameters. Both PULΔN5 and PULΔN106 gave higher production levels of protein than the wide type and displayed increased specific activities. Kinetic studies showed that substrate affinities of the mutants were improved in various degrees and the catalytic efficiency of PULΔN5, PULΔN45, PULΔN78, PULΔN106 and PULΔC9 were enhanced. However, the truncated mutations did not change the advantageous properties of the enzyme involving optimum temperature and pH for further application. Therefore, Disorder prediction-based truncation would be helpful to efficiently improve the enzyme activity and catalytic efficiency.

Auto-induction-based rapid evaluation of extracellular enzyme expression from lac operator-involved recombinant Escherichia coli.

Degeneration of engineered strains and decreased production of target gene products were often observed during recombinant bioprocess. Although several strategies have been developed to ensure high levels of target gene products, these methods seemed to be complex and laborious. By investigating possible factors contributing to the decreased yield, degeneration of host cells was identified as the main cause. Based on the principle of auto-induction and the fact that the interaction between colored substrates and gene products could present visible changes, a convenient and accurate screening method was developed to evaluate the production performance of engineered strains. Due to the typicality of pullulanase from genus Klebsiella, which has been a model for the research of secretion mechanism in gram-negative bacteria, an engineered E. coli producing extracellular pullulanase was employed to illustrate the method. Consequently, according to the capability to form a colorless halo, colonies could be divided into two groups, one surrounded by clear zone and the other unable to present transparent haloes. Furthermore, the high capability of these colonies was confirmed by performing pullulanase production in liquid medium. Compared with other methods for evaluating engineered strains, the visible screening technique was suggested to have the advantages of effectiveness and accuracy.