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

Alexander Grosse-Honebrink,Ying Zhang,Zak Bean,Gareth T Little,Edward Green,Nigel P Minton,Dana Heldt,Ruth H M Cornock,Klaus Winzer

doi:10.3389/fbioe.2021.659895

Abstract

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

Highlights

Active compounds are much sought after by the chemical and pharmaceutical industry
Based on previous studies in E. coli (Kataoka et al, 2013), a pathway to chirally pure (R)-1,3-BDO in C. saccharoperbutylacetonicum was formulated based on the conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA using the C. necator enzyme acetoacetyl-CoA reductase encoded by phaB
In E. coli the condensation of two acetyl-CoA to one acetoacetyl-CoA was reliant on the over-expression of the C. necator phaA gene

Summary

Introduction

Active compounds are much sought after by the chemical and pharmaceutical industry. 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)

Methods

Results

Conclusion