1. Introduction
Succinic acid recently garnered increasing attention as a promising alternative biochemical to replace petroleum-derived compounds due to its wide range of potential industrial application in the fields of pharmaceuticals, agriculture, and the food industry among others. Notably, succinic acid has been ranked as the most valuable chemical among the top 12 bio-based high-value-added chemicals according to the US Department of Energy (DOE).[1] Succinic acid, which belonging to the four-carbon dicarboxylic family, is an intermediate of the citric acid or glyoxylate cycle during glucose metabolism. Several microbes such as Actinobacillus succinogenes ,Mannheimia succiniciproducens , Escherichia coli ,Yarrowia lipolytica , and Corynebacterium glutamicum are known to produce succinic acid through anaerobic fermentation.[2–5]
C. glutamicum is a gram-positive soil bacterium that has been used for production of amino acids and other value-added metabolites. Succinic acid and lactic acid are the main excreted metabolites in the glucose metabolic pathway of wild-type C. glutamicum under anaerobic conditions, and the synthesis of succinic acid increases when the production of lactic acid is disrupted by the knock-out of lactate dehydrogenase 1 gene.[6] A previous study characterized the succinic acid transporter (SucE) of C. glutamicum with the authors reporting that, unlike the Dcu family of succinic acid transporters present in E. coli , the exported succinic acid was not imported from the medium.[7]The transcription level of sucE gene under anaerobic condition was also determined to be 2.2–fold higher than under aerobic conditions.[8] A metabolically engineered C. glutamicum strain (lactate dehydrogenase 1 knock-out (ΔldhA ) and over-expression of pyruvate carboxylate (pyc ) was reported to produce up to 146.4 g L-1 of succinic acid in a fed-batch condition from glucose alone, within 46 h, and under anaerobic conditions.[5] These results highlight the promising potential of C. glutamicum as a succinic acid producer. However, there is a lack of intensive research on succinic acid production from lignocellulosic biomass containing with glucose, xylose, arabinose, mannose and other components.
Classic homologous recombination has been developed to generate site-directed mutagenesis, gene deletion, simultaneous heterogeneous gene expressions and a homogeneous target gene knock-out in the C. glutamicum genome; moreover, this approach has been applied to enhance the production of amino acids and metabolites.[9-11] However, the efficiency of homologous recombination is known to be extremely low during the first and second crossover gene recombination, thus, extensive PCR screening must first be conducted to identify the desired recombinant colonies.
The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) genome editing system has been developed to provide simple and precise nucleotide editing, specific-gene deletions, and heterogeneous gene insertions into the genomic DNA of microbial, yeast, and human cells.[12–14] However, CRISPR-Cas9 or dCas9 (deactivated Cas9) cannot be used to edit the genome of C. glutamicum due to the toxic metabolites secreted by this microbe. In contrast, Cpf1, which was identified as a single-strand RNA-guided endonuclease belonging to the class 2 CRISPR-Cas system, was instead found to efficiently achieve these nucleotide substitutions, insertions, and deletions in C. glutamicum .[15]Therefore, CRISPR/Cpf1 genome editing can be used to reinforce succinic acid production through the precise deletion of the ldhA target gene and promote the expression of the genes of interest in C. glutamicum .
Lignocellulosic biomass has been evaluated as a sustainable and alternative sugar resource to corn-based sugar due to its abundance and high amount of carbohydrates.[16] Furthermore, biorefineries are considered environmentally green facilities that can potentially replace petroleum-based industries by using convertible sugars obtained from biomass in the fermentation process to produce biochemicals such as bioethanol, lactic acid, and succinic acid.[17–19] However, the structural complexity of lignocellulosic biomass enforces the highest cost input to the pretreatment and enzymatic hydrolysis in the overall bioconversion process.
Softwood is composed of ray parenchyma cells, resin canals, and tracheids. Among these structures, tracheids account for approximately 91% of the softwood xylem.[20] The macro- and microfibril structures of tracheids, and their composition of lignin, hemicellulose, and cellulose affect the pretreatment efficiency, although the extent to which depends on the method and condition. Woody plants including pine wood are more recalcitrant than herbaceous agricultural biomass such as corn stover, kenaf, rapeseed straw, and rice straw, when they are pretreated under popping and steam explosion.[21–25] Organosolv and dilute acid techniques are more effective for the hydrolysis of hardwoods (poplar and eucalyptus) than softwoods (pine and spruce).[16, 25] Sulfite pretreatment on aspen, eucalyptus, spruce, and red pine led to efficient hydrolysis rates.[26, 27] HPAC pretreatment conducted in this study, delignification of pine wood with hydrogen peroxide and acetic acid, removes lignin disturbances on cellulase and reduces cellulose recalcitrance, resulting in the highly efficient enzymatic digestion of pine wood.[28]
Succinic acid has been produced using metabolically engineered C. glutamicum and pure glucose as a carbon source. However, few studies have explored the applicability of lignocellulosic biomass, including corn cobs hydrolysate[29], for the production of succinic acid. Lignocellulosic biomass must first be pretreated prior to its saccharification and fermentation. Toxic by-products derived from lignin or hemicellulose are released during its pretreatment and are known to hinder the fermentation efficiency of the microbes.[30] Although few studies have assessed the applicability of lignocellulosic biomass and the value it added to the bioconversion process, the fermentation of hydrolysates from lignocellulosic biomass is expected to differ from that of pure glucose. Therefore, our study sought to characterize the production of succinic acid from softwood and analyze the patterns of succinic acid and the other metabolites produced by a CRISPR/Cpf1 generated ldhA mutant (ΔldhA-6 ), and aimed to enhance succinic acid production in a fed-batch system by utilizing a co-expression transformant (Psod:sucE-ΔldhA ).