Production of 1,5-diaminopentane and succinate in a single
Corynebacterium glutamicum strain via a bidirectional
metabolic switch controlled by the oxygen level
Yota TSUGE
Institute for Frontier Science Initiative, Kanazawa University
ReportIn this study, we successfully constructed a Corynebacterium glutamicum strain that can
produce 1,5-diaminopentane under aerobic conditions and succinate under anaerobic
conditions by metabolic engineering. In this strain, the oxygen level controls a bidirectional
metabolic switch, which channels the carbon flow into different metabolic pathways.
Research aims
With the growing concern over global warming, the bio-production of polyamide
monomers has attracted increasing attention in recent years. C. glutamicum, which is mainly
known as an amino acid producer, has been used as a host strain capable of producing thepolyamide monomers 1,5-diaminopentane and succinate[1-2]. This study aimed to construct aC. glutamicum strain that can produce 1,5-diaminopentane under aerobic conditions, andsuccinate under anaerobic conditions. In this strain, changes in the oxygen level can trigger ametabolic switch directing the carbon flow between the Embden-Meyerhof pathway (EMP)
and the Pentose Phosphate pathways (PPP).
Methods
The C. glutamicum ATCC 13032 strain was genetically engineered to construct a strain
that produces, as the main product, 1,5-diaminopentane and succinate under aerobic andanaerobic conditions, respectively. In the parental strain used for this study, the genes
encoding lactate dehydrogenase, menaquinone oxidoreductase, acetyl-CoA transferase,
acetate kinase, and phosphotransacetylase were inactivated to minimize the production oflactate and acetate as by-products under anaerobic conditions. Moreover, the lysine
carboxylase gene from Escherichia coli was introduced in the genome of C. glutamicum topromote the biosynthesis of 1,5-diaminopentane from lysine. A single mutation in theaspartokinase gene was also introduced to prevent product inhibition by lysine. Furthermore,
to facilitate the accumulation of oxaloacetate, a precursor of both 1,5-diaminopentane andsuccinate, a single mutation was introduced in the pyruvate carboxylase gene, which wasoverexpressed. We further engineered this parental strain to shut off the carbon flux into theEMP under aerobic conditions by replacing the promoter of the glucose-6-phosphate
isomerase (pgi) gene with the lactate dehydrogenase (ldhA) promoter. The constructed strain
was grown in minimal medium to produce 1,5-diaminopentane under aerobic conditions. For
succinate production, the strain was first grown in nutrient rich medium, and then the cells
were washed with minimal medium, followed by preparation of 6% of cell suspension toproduce succinate under anaerobic conditions. The products were analyzed byhigh-performance liquid chromatography (HPLC).
Results
Under aerobic conditions, four moles of NADPH are required to synthesize one mole of1,5-diaminopentane. Since the PPP is the primary source of NADPH production, ideally, all
the glucose carbons should be metabolized via this pathway. To that end, we constructed apromoter exchange strain by replacing the native promoter of the pgi gene with the promoterof the ldhA gene, which is induced under oxygen-deprived conditions. Hence, under aerobic
conditions, pgi expression is shut off and glucose is metabolized only via the PPP (Fig. 1). Incontrast, under anaerobic conditions, pgi expression is induced, channeling the glucose flux
into the EMP to produce succinate through pyruvate (Fig. 1).

Fig. 1 Schematic of the oxygen level controlled bidirectional metabolic switch between
the Embden-Meyerhof and Pentose Phosphate pathways.
Compared to the parental strain, the promoter exchange strain produced 1,5-diaminopentane
with a 4.1-fold and 2.3-fold higher yield (g per g of consumed glucose) and productivity(g/L·h), respectively (Fig. 2). However, compared to the parental strain, the succinate
productivity was decreased by 27% in the promoter exchange strain (Fig. 3). To resolve thisunexpected decrease in succinate productivity, we introduced in the genome of the promoter
exchange strain, an additional copy of the pgi gene under the control of the ldhA promoter.
In the resulting second copy strain, the succinate productivity and yield were comparable tothose in the parental strain (Fig. 2). Moreover, although the second copy of the pgi cassettedecreased the 1,5-diaminopentane yield by 27%, this strain exhibited an increased growthrate, resulting in a 10% increase in the 1,5-diaminopentane productivity compared to that in
the promoter exchange strain (Fig. 2).

Fig. 2 1,5-Diaminopentane productivity ( ) and yield () under aerobic conditions.

Fig. 3 Succinate productivity ( ) and yield ( ) under anaerobic conditions.

Conclusion
Using oxygen as a metabolic switch, two polyamide monomers were produced by a single
C. glutamicum strain through switching the carbon flow between the central metabolic
pathways. This strategy could be applied to produce other useful bio-chemicals and fuels.

References
1) Kind S. et al. (2014) From zero to hero – Production of bio-based nylon from renewable
resources using engineered Corynebacterium glutamicum. Metab. Eng. 25: 113–123.
2) Okino S., Noburyu R., Suda M., Jojima T., Inui M., Yukawa H. (2008) An efficientsuccinic acid production process in a metabolically engineered Corynebacterium
glutamicum strain. Appl. Microbiol. Biotechnol. 81: 459–464.