Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens

Abstract

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.

Central pathway engineering for enhanced succinate biosynthesis from acetate in Escherichia coli

“…Meanwhile, the availability of intracellular NADH can be increased through overexpressing a heterologous NAD + ‐dependent formate dehydrogenase (FDH) from Candida boidinii , which can converts formate to CO 2 and NADH (Balzer, Thakker, Bennett, & San, ; Berrı́os‐Rivera, Bennett, & San, ). Formate can be used as an alternative carbon and energy source using the FDH pathway due to CO 2 and NADH generation (Ahn, Bang, Kim, & Lee, ). In addition, the energy carrier, formate, also can be synthesized from CO 2 and H 2 (Li et al, ).…”

“…Formate can be used as an alternative carbon and energy source using the FDH pathway due to CO 2 and NADH generation (Ahn, Bang, Kim, & Lee, 2017). In addition, the energy carrier, formate, also can be synthesized from CO 2 and H 2 (Li et al, 2012).…”

Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase

“…The M. succiniciproducens PALK strain, which was developed by deleting lactate dehydrogenase (LDHA), phosphate acetyltransferase (PTA), and acetate kinase (ACKA) to prevent byproduct formation, produced nearly homo-SA with high productivity 15 . Moreover, various studies including the use of inexpensive carbon sources 16 , membrane engineering for strain robustness 17 , and development of effective fermentation 9 and downstream processes 15 were carried out. However, there had been no effort exerted to improve the enzymes to further improve SA production.…”

Bio‐based succinic acid: an overview of strain development, substrate utilization, and downstream purification

“…On the basis of the above strain LPK7, fdh gene from Methylobacterium extorquens was expressed to enhance formic acid assimilation when using glucose, glycerol, or sucrose as a primary carbon source. As a result, 76.1 g L −1 of succinic acid was produced at the end of fed‐batch fermentation using co‐substrate of sucrose and formic acid . Elementary mode analysis accompanied by clustering and membrane engineering was also successfully carried out in M. succiniciproducens for the purpose of enhancing the succinic acid production capacity and pH tolerance, respectively …”