Characterization of a non-phosphotransferase system for cis,cis-muconic acid production in Corynebacterium glutamicum

Abstract

Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.

Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

“…It has been shown that 3,4-DHBA displays antibacterial [ 3 ], antimutagenic [ 4 ], anti-inflammatory [ 5 ], antihyperglycemic [ 6 ] and highly antioxidant [ 7 ] effects. It also has potential applications for further microbiological synthesis of numerous valuable compounds, including the bioplastic precursor cis , cis -muconic acid [ 8 , 9 ]. To produce 3,4-DHBA from glucose, the synthesis of this compound from DHS, an intermediate in the common aromatic pathway, was previously implemented in E .…”

Metabolic engineering of Corynebacterium glutamicum for the production of cis, cis-muconic acid from lignin

“…Different microorganisms, such as Pseudomonas putida KT2440 [ 4 ], Amycolatopsis species ATCC 39116 [ 15 ], and E. coli [ 5 ] have been engineered to produce MA either via biosynthesis from glucose [ 6 – 8 ] and glycerol [ 2 ] or via biotransformation from aromatics [ 9 – 11 ]. The latter is particularly advantageous, because it requires only a few biochemical reactions and offers molar yields up to 100% [ 4 ].…”

“…In contrast to aromatics-based biotransformation, de-novo biosynthesis of MA from glucose to glycerol is less efficient. So far, MA titer (5 g L −1 ) and productivity (0.03 g L −1 h −1 ) have remained rather low [ 2 , 7 , 8 ].…”

Corynebacterium Cell Factory Design and Culture Process Optimization for Muconic Acid Biosynthesis

“…A variety of engineering strategies to increase intracellular levels of PEP (phosphoenolpyruvate) and E4P (erythrose 4-phosphate), key intermediates of the shikimate pathway, and additional engineering to efficiently utilize various carbon sources including glucose, will lead us to obtain more efficient MA-producing Corynebacterium cell factories. Recently further engineered strain with deletion of the iolR gene which greatly increased glucose uptake has been developed, which had non-phosphotransferase system with increased PEP availability, showing improved MA production yield in the flask culture 55 .…”