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S-Substituted-l-cysteine sulfoxides are valuable compounds that are contained in plants. Particularly, (+)-alliin and its degraded products have gained significant attention because of their human health benefits. However, (+)-alliin production has been limited to extraction from plants and chemical synthesis; both methods have drawbacks in terms of stability and safety. Here, we proposed the enzymatic cascade reaction for synthesizing (+)-alliin from readily available substrates. To achieve a one-pot (+)-alliin production, we constructed Escherichia coli coexpressing the genes encoding tryptophan synthase from Aeromonas hydrophila ssp. hydrophila NBRC 3820 and l - isoleucine hydroxylase from Bacillus thuringiensis 2e2 for the biocatalyst. Deletion of tryptophanase gene in E. coli increased the yield about 2-fold. Under optimized conditions, (+)-alliin accumulation reached 110 mM, which is the highest productivity thus far. Moreover, natural and unnatural S-substituted-l-cysteine sulfoxides were synthesized by applying various thiols to the cascade reaction. These results indicate that the developed bioprocess would enable the supply of diverse S-substituted-l-cysteine sulfoxides.

A wide variety of S-substituted cysteine derivatives occur in plant metabolites. For example, S-allyl-l-cysteine (SAC), mainly contained in garlic, gathers huge interest because of its favorable bioactivities for human health. However, conventional methods for preparing SAC suffer from several drawbacks with regard to efficiency and toxicity, which highlights the need for improved processes for SAC synthesis. This study aims to develop a novel bioprocess to produce SAC by microbial enzymes from easily available substrates. We found that Escherichia coli had the ability to synthesize SAC from allyl mercaptan, pyruvic acid, and ammonium sulfate. An enzyme purification through 3-step column chromatography, followed by determination of the N-terminal amino acid sequence revealed that tryptophanase (TnaA) was the enzyme responsible for SAC formation. Although the enzyme catalyzed the reversible reaction for synthesizing and degrading SAC, the degradation proceeded significantly faster than the synthesis. Interestingly, TnaA catalyzed the synthesis of a wide range of S-substituted cysteines with alkyl chains or aromatic rings, some of which are present in Allium and Petiveria plants. Our results showed a novel substrate specificity of TnaA toward various S-substituted cysteine. TnaA is a promising biocatalyst for developing a new process to supply various valuable S-substituted cysteine derivatives for medicinal and health-promoting applications., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway., Results: Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2'-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2'-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2'-deoxyinosine, the deaminated product of 2'-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process., Conclusion: A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses.

The stereo-specific L-isoleucine-4-hydroxylase (L-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to "shunt" destroyed TCA, thereby coupling L-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of L-isoleucine into 4-HIL with an 82% yield.

Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain. In the reaction with recombinant Escherichia coli expressing P450nov, FDXnov, and FDRnov, BMAL-OH was successfully produced from BMAL. Moreover, using the directed evolution approach, four amino acid substitutions, L188P/F218L/L237M in P450nov and A10T in FDXnov, were found to enhance BMAL-OH production. Consequently, up to 5.2 g/L BMAL-OH was obtained from 8.0 g/L BMAL by bioconversion using a 250-mL jar fermenter, indicating that this strain may be useful for synthesis of maltol derivatives which could have potential applications in various fields. [ABSTRACT FROM AUTHOR]

Transcriptome data for a xylitol-producing recombinant Escherichia coli were obtained and used to tune up its productivity. Structural genes of NADPH-dependent D-xyloSe reductase and D-xylose permease were inserted into an Escherichia coli chromosome to construct a recombinant strain producing xylitol from n-xylose for use as a model system for NADPH-dependent bioconversion. Transcriptome analysis of xylitol-producing and nonproducing conditions for the recombinant revealed that xylitol production down-regulated 56 genes. These genes were then selected as candidate factors for suppression of the NADPH supply and were disrupted to validate their functions. Of the gene disruptants, that resulting from the deletion of yhbC showed the best bioconversion rate. Also, the deletion accelerated cell growth during log phase. The features of the mutant could be maintained in jar fermenter-scale production of xylitol. Thus, our novel molecular host strain breeding method using transcriptome analysis was fully effective and could be applied to improving various industrial strains. [ABSTRACT FROM AUTHOR]