Enhancement of sucrose metabolism in Clostridium saccharoperbutylacetonicum N1-4 through metabolic engineering for improved acetone–butanol–ethanol (ABE) fermentation

Highlights

• PTS system is the primary sucrose catabolic pathway in C. saccharoperbutylacetonicum.

• Colloid-like polysaccharide was generated after inactivation of sucrose operon.

• Deletion of scrR alleviated CCR effect and enhanced sucrose consumption.

• Overexpressing native sucrose pathway enhanced sucrose utilization & ABE production.

• Sugarcane juice can be used as an inexpensive substrate for ABE fermentation.

Abstract

This work investigated sucrose metabolism in C. saccharoperbutylacetonicum. Inactivation of sucrose catabolism operon resulted in 28.9% decrease in sucrose consumption and 44.1% decrease in ABE production with sucrose as sole carbon source. Interestingly, a large amount of colloid-like polysaccharides were generated in the mutant, which might be due to inefficient intracellular sucrose metabolism. Deletion of transcriptional repressor gene successfully alleviated CCR and enhanced ABE production by 24.7%. Additional overexpression of endogenous sucrose pathway further elevated sucrose consumption and enhanced ABE production by 17.2%, 45.7%, or 22.5% compared to wild type with sucrose, mixed sugars or sugarcane juice as substrate, respectively. The engineered strain could be a robust platform for efficient biofuel production from inexpensive sucrose-based carbon sources.

Introduction

Biofuels and biochemicals produced from renewable and inexpensive carbon sources have great potential to address the global problems as related to the exhaustion of the conventional fossil fuels (Chen et al., 2013, Kudahettige-Nilsson et al., 2015, Munasinghe and Khanal, 2010, Zhang et al., 2016). Biobutanol produced through the acetone-butanol-ethanol (ABE) fermentation with solventogenic clostridia has been of great interests because it can be either used as a fuel source or as a valuable chemical feedstock for various industries (Lee et al., 2016, Ren et al., 2016). ABE fermentation has a history of >100 years for solvent production and strains from various solventogenic Clostridium species have been evaluated for ABE production, including Clostridium acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum (Green, 2011, Moon et al., 2016). Among them, C. saccharoperbutylacetonicum is highlighted by its high butanol selectivity (73–85% of the total ABE produced) and low sporulation frequency (beneficial for the industrial fermentation) (Keis et al., 2001). Recently, high-efficiency transformation protocols and CRISPR-Cas9-based genome editing tools have been developed for C. saccharoperbutylacetonicum (Herman et al., 2017, Wang et al., 2017), enabling great potential to further develop this organism as a chassis strain for industrial ABE production. Previously, numerous renewable feedstocks have been tested for ABE fermentation using C. saccharoperbutylacetonicum, including industrial wastes (Hipolito et al., 2008, Mun et al., 1995), cassava (Thang et al., 2010), starch (Al-Shorgani et al., 2012b, Thang and Kobayashi, 2014), wastewater algae (Castro et al., 2015, Ellis et al., 2012), rice bran (Al-Shorgani et al., 2012a), lignocellulosic hydrolysate (Chen et al., 2013, Zheng et al., 2015), and palm kernel cake (Shukor et al., 2016, Shukor et al., 2014).

Sucrose is one of the most abundant, readily available and inexpensive carbon source that can be obtained from sugarcane and sugarbeet. It can be used as a promising substrate for cost-effective production of biofuels and bioproducts. Generally, there are three types of sucrose metabolism pathways in bacteria responsible for sucrose transportation and utilization (Reid and Abratt, 2005, Sahin-Tóth et al., 2000). The phosphotransferase system (PTS) is the major mechanism for sucrose metabolism in bacteria, generally containing the phosphoenolpyruvate (PEP)-dependent sucrose-specific phosphotransferase, sucrose-6-phosphate hydrolase, and ATP-dependent fructokinase. In addition to PTS, there are two non-PTS systems have been elucidated for sucrose metabolism in bacteria; one is comprised of the sucrose permease in combination with the sucrase, and the other consists of the sucrose permease in combination with the sucrose phosphorylase. Usually, the genes involved in each of the three sucrose metabolism systems as discussed above are clustered in a polycistronic operon.

Previously, the sucrose metabolism in various solventogenic clostridial strains has been investigated by several researchers. A sucrose operon was identified in C. beijerinckii NCIMB 8052, containing three genes (encoding a sucrose-specific enzyme IIBC protein of PTS, a sucrose hydrolase, and a fructokinase, respectively) involved in the sucrose PTS and a transcriptional repressor gene which could control the transcription of the whole operon (Fig. 1a) (Reid et al., 1999, Tangney et al., 1998). Disruption of this operon resulted in a strain unable to metabolize sucrose, suggesting that PTS was the only sucrose catabolic pathway in C. beijerinckii NCIMB 8052 (Reid et al., 1999). In C. acetobutylicum ATCC 824, a similar sucrose metabolic pathway as that in C. beijerinckii NCIMB 8052 was reported (Tangney and Mitchell, 2000). Although the genes involved in the pathway were conserved, the organization and regulation of the operon in C. acetobutylicum was remarkably different from that in C. beijerinckii (Fig. 1a).

C. saccharoperbutylacetonicum can naturally utilize sucrose. However, Ogata et al. reported that when exponentially growing cells of this microorganism were exposed to high concentrations of sucrose (0.3–0.5 M), the cell morphology was notably changed and sucrose-induced cell autolysis was observed (Ogata et al., 1980). Generally, the sucrose metabolism in C. saccharoperbutylacetonicum is not well understood. Therefore, in this study, for the first time, the primary sucrose catabolic pathway in C. saccharoperbutylacetonicum N1-4 was identified and characterized through gene deletion using the CRISPR-Cas9 system. In addition, the sucrose consumption and ABE production in C. saccharoperbutylacetonicum were further enhanced by deleting the transcriptional repressor gene and overexpressing the endogenous sucrose catabolic pathway. Finally, a robust strain capable of efficient sucrose consumption for enhanced ABE production was developed, and potential viable biofuel production from inexpensive sucrose-based carbon sources was demonstrated.