The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid.

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid-enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2-oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three-enzyme co-expression system containing L-threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5 M L-threonine could be converted to L-2-ABA with a 99% molar conversion rate and a space-time yield of 51.5 g·L-1 ·h-1 . In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.

A novel NADH-dependent leucine dehydrogenase for multi-step cascade synthesis of L-phosphinothricin.

L-Phosphinothricin (L-PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L-PPT has always been the focus in pesticide industry. In recent years, "one-pot, two-step" enzyme-mediated cascade strategy is a mainstream pathway to obtain L-PPT. Herein, RgDAAO and BsLeuDH were applied to expand "one-pot, two-step" process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis (BsLeuDH) was firstly characterized and attempted to generate L-PPT, achieving an excellent enantioselectivity (99.9% ee). Meanwhile, a formate dehydrogenase from Pichia pastoris (PpFDH) was utilized to implement NADH cofactor regeneration and only CO2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D-PPT relying on a D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) with content conversion (46.1%). L-PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by RgDAAO and reductive amination catalyzed by BsLeuDH, achieving 80.3% overall yield and > 99.9% ee value.

Rational identification of a high catalytic efficiency leucine dehydrogenase and process development for efficient synthesis of l-phenylglycine.

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l-phenylglycine (l-Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8 U mg-1 . Through reaction process optimization, a high-concentration substrate (0.7 m) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 m BFA could be aminated to l-Phg with conversion of >99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg.

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.

Efficient single whole-cell biotransformation for L-2-aminobutyric acid production through engineering of leucine dehydrogenase combined with expression regulation.

Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA, EsldhK72A, fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3ivlA-EsldhK72A-fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L-1·h-1, which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

[Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid].

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid / 生物工程学报

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L-1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L-1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L-1 to 2.72 ± 0.11 g·L-1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L-1 l-leucine with a glucose conversion efficiency of 0.191 g·g-1.

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178.

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD⁺ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L-1 min-1 g-1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L-1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L-1 dry cells and 50.4 g L-1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.

Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production.

L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD+ regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The "best" variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency (k cat/K M) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L-ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.

Establishing a Mathematical Equations and Improving the Production of L-tert-Leucine by Uniform Design and Regression Analysis.

L-tert-Leucine (L-Tle) and its derivatives are extensively used as crucial building blocks for chiral auxiliaries, pharmaceutically active ingredients, and ligands. Combining with formate dehydrogenase (FDH) for regenerating the expensive coenzyme NADH, leucine dehydrogenase (LeuDH) is continually used for synthesizing L-Tle from α-keto acid. A multilevel factorial experimental design was executed for research of this system. In this work, an efficient optimization method for improving the productivity of L-Tle was developed. And the mathematical model between different fermentation conditions and L-Tle yield was also determined in the form of the equation by using uniform design and regression analysis. The multivariate regression equation was conveniently implemented in water, with a space time yield of 505.9 g L-1 day-1 and an enantiomeric excess value of >99 %. These results demonstrated that this method might become an ideal protocol for industrial production of chiral compounds and unnatural amino acids such as chiral drug intermediates.

Expression, purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile Laceyella sacchari.

OBJECTIVE: A potential thermotolerant L-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized. RESULTS: Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C. CONCLUSION: It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.

L-tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare L-tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the L-tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of L-tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies (k cat/K m) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of L-tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme.

[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.

A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes.

We created a novel chimeric amine dehydrogenase (AmDH) via domain shuffling of two parent AmDHs ('L- and F-AmDH'), which in turn had been generated from leucine and phenylalanine DH, respectively. Unlike the parent proteins, the chimeric AmDH ('cFL-AmDH') catalyzes the amination of acetophenone to (R)-methylbenzylamine and adamantylmethylketone to adamantylethylamine.

A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH-regeneration system based on L-leucine dehydrogenase and formate dehydrogenase.

L-2-Aminobutyric acid (L-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. It can be produced by transaminase or dehydrogenase from α-ketobutyric acid, which can be synthesized enzymatically from the bulk amino acid, L-threonine. Deamination of L-threonine followed by a hydrogenation reaction gave almost the theoretical yield and was estimated to be more cost-effective than the established chemical process. L-Threonine deaminase from Escherichia coli, L-leucine dehydrogenase from Bacillus cereus, and formate dehydrogenase from Pseudomonas sp. were over-expressed in E. coli and used for one-pot production of L-ABA with formate as a co-substrate for NADH regeneration. 30 mol L-threonine were converted to 29.2 mol L-ABA at 97.3 % of theoretical yield and with productivity of 6.37 g l(-1) h(-1) at 50 l. This process offers a promising approach to fulfil industrial requirements for L-ABA.

A combinatorial approach to detect coevolved amino acid networks in protein families of variable divergence.

Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.