Abstract
3-Hydroxypropanoic acid (3-HP) is an important biomass-derivable platform chemical that can be converted into a number of industrially relevant compounds. There have been several attempts to produce 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from K. pneumoniae. Genetic engineering, driven by in silico optimization, and optimization of cultivation conditions resulted in a 3-HP titer of 10 g/L, in a standard batch cultivation. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis. With this relatively high titer in batch, and the robustness of B. subtilis in high density fermentation conditions, we expect that our production strains may constitute a solid basis for commercial production of 3-HP.
Highlights
Concerns over usage of fossil fuels have led to the development of sustainable technologies to convert renewable materials into various compounds such as alcohols, acids, and chemicals (Hanai et al, 2007; Atsumi and Liao, 2008; Atsumi et al, 2008; McKenna and Nielsen, 2011; Borodina et al, 2015)
We examined the effect of 3-HPA and 3-Hydroxypropanoic acid (3-HP) on B. subtilis growing on the M9 minimum medium supplemented with glycerol
Bacillus subtilis has the potential to be considered as a microbial host for production of 3-HP due to several reasons including its efficient glycerol import system and high growth rate on glycerol which compared to E. coli is an advantage
Summary
Concerns over usage of fossil fuels have led to the development of sustainable technologies to convert renewable materials into various compounds such as alcohols, acids, and chemicals (Hanai et al, 2007; Atsumi and Liao, 2008; Atsumi et al, 2008; McKenna and Nielsen, 2011; Borodina et al, 2015). The second reaction is the conversion of 3-HPA to 3-HP, catalyzed by an aldehyde dehydrogenase (Raj et al, 2008) This biosynthetic pathway is present in some natural producers of 3-HP, such as Klebsiella pneumoniae (Kumar et al, 2012; Ashok et al, 2013) and Lactobacillus reuteri (patent: US 20070148749 A1). We explored the possibility to use and alternative host for heterologous expression of the 3-HP synthetic pathway, the Gram-positive model organism Bacillus subtilis It is non-pathogenic, generally recognized as safe (GRAS), suitable for large scale cultivation and has high growth rates. We expressed the 3-HP synthetic pathway from K. pneumoniae in B. subtilis Since this particular glycerol dehydratase is sensitive to oxygen, micro-aerobic/anaerobic condition were required for optimal 3-HP production (Zhao et al, 2015). We carried out several rounds of strain and medium optimization, which resulted in a production strain capable of producing 10 g/L of 3-HP in shaking flasks, with the yield on glycerol ranging from 70 to 80%
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