Redox self-sufficient whole cell biotransformation for amination of alcohols

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD⁺ was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best.     

Enantioselective synthesis of (S)-phenylephrine by recombinant Escherichia coli cells expressing the short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811

BackgroundAn amino alcohol dehydrogenase gene (RE_AADH) from Rhodococcus erythropolis BCRC 10909 has been used for the conversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-phenylephrine [(S)-PE]. However RE_AADH uses NADPH as cofactor, and only limited production of (S)-PE from HPMAE is achieved.MethodsA short-chain dehydrogenase/reductase gene (SQ_SDR) from Serratia quinivorans BCRC 14811 was expressed in Escherichia coli BL21 (DE3) for the conversion of HPMAE to (S)-PE.ResultsThe SQ_SDR enzyme was capable of converting HPMAE to (S)-PE in the presence of NADH and NADPH, with specific activities of 26.5 ± 2.3 U/mg protein and 0.24 ± 0.01 U/mg protein, respectively, at 30 °C and at a pH of 7.0. The E. coli BL21 (DE3), expressing NADH-preferring SQ_SDR, converted HPMAE to (S)-PE with more than 99% enantiomeric excess, a conversion yield of 86.6% and a productivity of 20.2 mmol/l h, which was much higher than our previous report using E. coli NovaBlue expressing NADPH-dependent RE_AADH as the biocatalyst.ConclusionThe SQ_SDR enzyme with its high catalytic activity and strong preference for NADH as a cofactor provided a significant advantage in bioreduction.     

酵母2-苯乙醇耐受性的研究进展

2-苯乙醇(2-phenylethanol, 2-PE)是一种可食用且有玫瑰香味的高级芳香醇,常用于食品、化妆品和药品行业。由于物理和化学法制备2-PE得率低,不适用于工业生产。而作为单细胞真核微生物的酵母具有高效合成“天然” 2-PE的潜力,因此酵母作为底盘微生物合成2-PE的策略深受研究者青睐。然而,在酵母进行2-PE发酵过程中不免会受到2-PE毒害作用影响。因此,亟须研究酵母耐受2-PE的机制为生产实际提供理论基础,这也有助于选育具有较高2-PE耐受性的酵母菌株。本文综述了酵母2-PE耐受性的研究进展,从酵母2-PE合成途径、2-PE耐受性机理等方面进行阐述,主要说明提升酵母2-PE耐受性的方法。掌握酵母2-PE耐受机制,最终提升酵母2-PE产量及转化效率是今后研究的重中之重。     

Effects of pyruvate dehydrogenase subunits overexpression on the α-ketoglutarate production in Yarrowia lipolytica WSH-Z06

Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Enantioselective synthesis of (S)-phenylephrine by whole cells of recombinant Escherichia coli expressing the amino alcohol dehydrogenase gene from Rhodococcus erythropolis BCRC 10909

(R)-phenylephrine [(R)-PE] is an α₁-adrenergic receptor agonist that is widely used in over-the-counter drugs to treat the common cold. We found that Rhodococcus erythropolis BCRC 10909 can convert detectable level of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE by high performance liquid chromatography tandem mass spectrometry analysis. An amino alcohol dehydrogenase gene (RE_AADH) which possesses the ability to convert HPMAE to (S)-PE was then isolated from R. erythropolis BCRC 10909 and expressed in Escherichia coli NovaBlue. The purified RE_AADH, tagged with 6×His, had a molecular mass of approximately 30 kDa and exhibited a specific activity of 0.19 μU/mg to HPMAE in the presence of NADPH, indicating this enzyme could be categorized as NADP⁺-dependent short-chain dehydrogenase reductase. E. coli NovaBlue cell expressing the RE_AADH gene was able to convert HPMAE to (S)-PE with more than 99% enantiomeric excess (ee), 78% yield and a productivity of 3.9 mmol (S)-PE/L h in 12 h at 30 °C and pH 7. The (S)-PE, recovered from reaction mixture by precipitation at pH 11.3, could be converted to (R)-PE (ee > 99%) by Walden inversion reaction. This is the first reported biocatalytic process for the production of (S)-PE from HPMAE.     

High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3′-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.     

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli

L -Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l -threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l -threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l -threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l -threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l -threonine production, we developed an l -glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02- and 1.21-fold increase in l -threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.     

生物转化法合成2-苯乙醇的研究进展

2-苯乙醇(2-phenylethanol,2-PE)是具有玫瑰香味的芳香醇,在食品、化妆品以及药品领域有着广泛的应用。但是从植物花卉中提取的天然2-苯乙醇产量低,成本高。目前,用微生物转化生产"天然"2-苯乙醇越来越受到关注。本文对近年来国内外报道的提高微生物转化合成2-苯乙醇产量的研究,尤其是生产菌株选育和发酵工程优化的研究进行了综述,并提出今后研究的新思路,旨在为利用微生物发酵进行2-苯乙醇生产提供参考。     

Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.     

Cloning Rosa hybrid phenylacetaldehyde synthase for the production of 2-phenylethanol in a whole cell Escherichia coli system

2-Phenylethanol (2-PE) is a desirable compound in the food and perfumery industries with a characteristic rose fragrance. Until now, most of the studied biotechnological processes to produce 2-PE were conducted using natural 2-PE-producing yeasts. Only several researches were conducted in other genetically engineered microorganisms that simulated the Ehrlich pathway for the conversion of amino acids to fusel alcohols. Here, a novel metabolic pathway has been designed in Escherichia coli to produce 2-PE, using the Rosa hybrid phenylacetaldehyde synthase (PAAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme capable of transforming l-phenylalanine (l-phe) into phenylacetaldehyde by decarboxylation and oxidation. To overcome the enzyme insolubility in E. coli, several plasmids and host strains were tested for their expression ability. The desired results were obtained by using the pTYB21 plasmid containing the intein tag from the Saccharomyces cerevisiae VMA1. It was discovered that the intein PAAS activity is temperature-dependent, working well in the range of 25 to 30 °C but losing most of its activity at 37 °C. When external PLP cofactor was added, the cells produced 0.39 g l^-1 2-PE directly from l-phe. In addition, a biotransformation that was based only on internal de novo PLP synthesis produced 0.34 g l^-1 2-PE, thus creating for the first time an E. coli strain that can produce 2-PE from l-phe without the need for exterior cofactor additions.     

Screening of Yeasts Isolated from Brazilian Environments for the 2-Phenylethanol (2-PE) Production

Phenylethanol alcohol, or 2-phenylethanol (2-PE) production by yeasts has been considered a promising alternative to its chemical synthesis. In order to evaluate the potential of yeast strains isolated from different Brazilian environments, we evaluated the 2-PE production of 267 strains. Among them, the Kluyveromyces marxianus CCT 7735 yeast stood out as being the best 2-PE producer. The K. marxianus CCT 7735 growth was impaired by 2-PE; nevertheless, this effect is less pronounced than the inhibition reported for certain Saccharomyces cerevisiae strains. The maximum 2-PE titer obtained under optimized conditions was 3.44 g/L, 28% higher than the titer achieved under unoptimized conditions. The optimized conditions were: 30ºC, and glucose and L-phe concentrations of 3.0 and 4.0 g/L, respectively. Moreover, the specific production rate of 2-PE increased twofold compared to the unoptimized conditions.     

High-yield whole cell biosynthesis of Nylon 12 monomer with self-sufficient supply of multiple cofactors

Biosynthesis of Nylon 12 monomer using dodecanoic acid (DDA) or its esters as the renewable feedstock typically involves ω-hydroxylation, oxidation and ω-amination. The dependence of hydroxylation and oxidation-catalyzing enzymes on redox cofactors, and the requirement of L-alanine as the co-substrate and pyridoxal 5′-phosphate (PLP) as the coenzyme for transamination, raise the issue of redox imbalance and cofactor shortage, challenging the development of efficient biocatalysts. Simultaneous regeneration of the redox equivalents, PLP and L-alanine required in the artificial pathway was enabled by its interfacing with the native metabolism of the host using glucose dehydrogenase (GDH), L-alanine dehydrogenase (AlaDH) and an exogenous ribose 5-phosphate (R5P)-dependent PLP synthesis pathway as bridges. Further engineering of the host by blocking β-oxidation and enhancing substrate uptake improved the ω-aminododecanoic acid (ω-AmDDA) yield to 96.5%. This study offers a strategy to resolve the cofactor imbalance issue commonly encountered in whole-cell biocatalysis and meanwhile lays a solid foundation for Nylon 12 bioproduction.     

Synergistic effect enhances 2-phenylethyl acetate production in the mixed fermentation of Hanseniaspora vineae and Saccharomyces cerevisiae

2-Phenylethyl acetate (2-PEA) is a desired aroma compound in wine due to its honey- and flowery-like characteristics. The effects of adding l-phenylalanine (Phe) during 2-PEA production were investigated in the co-fermentation of Hanseniaspora vineae (HV6) and Saccharomyces cerevisiae BDX. BDX and HV6 strains overproduced 2-phenylethyl alcohol (2-PE) and 2-PEA, respectively. The co-fermentation of BDX and HV6 achieved a 14.9 fold increase in 2-PEA odour activity value (OAV) but a 42.0 % reduction of 2-PE OAV compared to BDX fermentation; the 2-PEA concentration was significantly higher than the sum of BDX and HV6 pure fermentations. This suggests that BDX and HV6 have synergistic effects on 2-PEA formation in mixed culture. Adding 151.6 mg/L Phe enhanced the OAV of 2-PEA by 52.8 % compared to the control. The combination of Phe addition with the co-fermentation of S. cerevisiae and H. vineae is a potential way to increase 2-PEA production and improve wine aromatic quality.     

Regeneration of the nicotinamide cofactor using a mediator-free electrochemical method with a tin oxide electrode

Tin (IV) oxide was made using an anodization and annealing method and was used as a working electrode in an electrochemical cofactor regeneration reaction. This material was formed with a large surface area, and by changing the preparation conditions, it was possible to control the morphology. Tin oxide has redox properties similar to those of frequently used mediators required for electron transfer between cofactors and an electrode. Therefore, by using tin oxide as a novel electrode, mediator-free electrochemical cofactor regeneration may be possible. Oxidation and reduction of the nicotinamide cofactors, NAD(P)H and NAD(P)⁺, were carried out under various reaction conditions. The results showed a high efficiency for oxidizing NADH over a broad range of pH and temperatures. The oxidation tendency of NADPH was also observed, and it demonstrated a similar reaction tendency as NADH. When using a tin oxide electrode, NAD⁺ was readily reduced to NADH, though the efficiency of this reaction was lower than for NADH oxidation. Oxidation of 2-propanol to acetone was used as a model system using alcohol dehydrogenase and the cofactor regeneration system suggested in this study. The electroenzymatic reaction showed efficient regeneration of NADP⁺ without a mediator.     

酵母菌合成2-苯乙醇的研究进展

2-苯乙醇是一种具有令人愉悦的玫瑰风味的芳香醇,在食品、化妆品和药品等领域具有广泛的应用。本文对酵母菌合成2-苯乙醇的代谢途径及其调控过程、以及提高2-苯乙醇产量的国内外研究进展进行了综述,并对通过微生物转化法合成2-苯乙醇目前存在的不足及进一步研究方向进行了讨论。     

Differences in hepatic and metabolic changes after acute and chronic alcohol consumption.

Hepatic metabolism of ethanol to acetaldehyde by the alcohol dehydrogenase pathway is associated with the generation of reducing equivalents as NADH. Conversely, reducing equivalents are consumed when ethanol oxidation is catalyzed by the NADPH dependent microsomal ethanol oxidizing system. Since the major fraction of ethanol metabolism proceeds via alcohol dehydrogenase and since the oxidation of acetaldehyde also generates NADH, an excess of reducing equivalents is produced. This explains a variety of effects following acute ethanol administration, including hyperlactacidemia, hyperuricemia, enhanced lipogenesis and depressed lipid oxidation. To the extent that ethanol is oxidized by the alternate microsomal ethanol oxidizing system pathway, it slows the metabolism of other microsomal substrates. Following chronic ethanol consumption, adaptive microsomal changes prevail, which include enhanced ethanol and drug metabolism, and increased lipoprotein production. Severe hepatic lesions (alcoholic hepatitis and cirrhosis) develop after prolonged ethanol consumption in baboons. These injurious alterations are not prevented by nutritionally adequate diets and can therefore be ascribed to ethanol rather than to dietary inadequacy.     

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner–Doudoroff pathway

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC^TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD⁺ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.