Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162

Abstract

Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.

Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH

“…1) Because beta-lactam antibiotics are the most commonly used antibacterial agents, the demand for (R)-1,3-BD has increased, and as a result, methods for (R)-1,3-BD production have been studied. 2,3) Thus far, 1,3-BD has been produced industrially in multistep chemical reactions as a racemic mixture of R and S forms, mostly from acetaldehyde prepared synthetically from fossil fuels. 4,5) In contrast, optically active 1,3-BD has been biologically synthesized from petroleum-based chemicals, such as 4-hydroxybutanone or its racemic and enantiomeric compounds, through microbial stereo-selective reduction or optical resolution.…”

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

“…Titres reached are similar to studies using plasmid borne expression of pyruvate decarboxylase from Zymomonas mobilis and deoxyribose-5-phosphate aldolase from Bacillus halodurans ( Nemr et al, 2018 ; Kim et al, 2020 ) but lower than in studies using plasmid borne expression of PhaA and PhaB from R. eutropha coupled with expression of Bld from C. saccharoperbutylacetonicum ( Kataoka et al, 2013 , 2014 ) summarised in Table 1 . Higher titres can be reached using 4-hydroxy-2-butanone as substrate ( Itoh et al, 2007 ; Zheng et al, 2012 ; Yang et al, 2014 ), however glucose is more widely available and more ecenomical than 4-hydroxy-2-butanone. Finally, future work should aim at optimising the fermentation and at upscaling of the production which is easier with an anaerobic organism such as the one used in this work.…”

“…Reduction through yeast fermentation of 4H2B led to low product yields when the desired stereo-specificity was reached ( Matsuyama et al, 2001 ). However, later research showed promising results when reducing 4H2B using either newly isolated yeast strains, with up to 100% enantiomeric excess and a titre of 38.2 g/l ( Zheng et al, 2012 ; Yang et al, 2014 ), or genetically engineered E. coli , where a 99% enantiomeric excess and 99% substrate yield were obtained ( Itoh et al, 2007 ). Another approach using genetically modified E. coli based on the enantio-selective oxidation of ( S )-1,3-BDO was initially promising ( Matsuyama et al, 2001 ) but proved difficult to scale-up ( Yamamoto et al, 2002 ).…”

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms

“…The specific applications of these metabolic engineering tools in microbial pathways are presented in Figure 3B. focused on the biotransformation of 4-hydroxy-2-butanone [123][124][125] or 1,3-BDO racemic mixture [126][127][128] to (R)-1,3-BDO. More recently, two artificial metabolic pathways for (R)-1,3-BDO production from glucose have been developed: (i) the reversed fatty acid β-oxidation pathway [129][130][131] and (ii) the 2-deoxyribose-5-phosphate aldolasealdo-keto reductase (DERA-AKR) pathway.…”