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 ).