Metabolic engineering of coryneform bacteria for the enhanced
supply of acetyl-CoA and its application to the fermentative
production of useful compounds
Naoya KATAOKA
Graduate School of Sciences and Technology for Innovation,
Yamaguchi University

Research aims
Corynebacterium glutamicum is a biotin auxotrophic Gram-positive soil bacterium andhas been used for the industrial production of amino acids, particularly glutamate and lysine.
Recent progress in metabolic engineering
strategies has enabled the development of C.
glutamicum strains capable of producing a varietyof useful compounds, including isobutanol,
itaconate, and cadaverine. However, little progresshas been made in the production of compounds
derived from acetyl coenzyme A (acetyl-CoA), an
essential intermediate of the central metabolism ofC. glutamicum. The present study aimed to
identify factors involved in increasing the pool ofacetyl-CoA (using genetic engineering tomodulate the expression of genes related to thesupply and consumption of acetyl-CoA) and toapply this knowledge to the production ofacetyl-CoA-derived compounds.
Methods
Using C. glutamicum ATCC 13032 as a parental strain, mutant strains with in-framedeletions of ramA, ldhA, pqo, cat, and pta-ackA were generated via a two-step homologous
recombination procedure. All C. glutamicum strains were grown in minimal medium
supplemented with 222 mM glucose to analyze their fermentation profiles.

Results
1. Effects of ramA deletion on the glucose metabolism of C. glutamicum
One effective way to enhance the supply of acetyl-CoA is to decrease the carbon flow into
the tricarboxylic acid (TCA) cycle. RamA, a global transcriptional regulator, directlyregulates many genes involved in the central metabolism of C. glutamicum (Fig. 1),
particularly those involved in the TCA cycle[1]. Compared to the wild type, when the ΔramA
strain was grown in a minimal medium supplemented with glucose, the expression of thegenes encoding CS, ACN, ODHC, SCS, SDH, FUM, and MQO was down-regulated.
Notably, the ΔramA strain showed approximately 90% less CS activity than the wild type[2].
Hence, the effects of ramA deletion on glucose metabolism were investigated further.
Compared to the wild type, the ΔramA strain showed an increased glucose consumption rate,
decreased growth, a higher formation of lactate and acetate, and lower accumulation of2-oxoglutarate and succinate (Table 1). These results indicate a reduction of the aerobic
metabolism (such as the TCA cycle) and an increase of fermentation in the ΔramA strain.
However, the specific activity of PDHC, the enzyme producing the acetyl-CoA, in theΔramA strain was significantly lower than in the wild type (4.40 mU·(mg of protein)-1 vs.
13.5 mU·(mg of protein)-1). Unfortunately, this finding is not in line with the aims of thisstudy. Therefore, new approaches need to be considered, such as (i) investigating the newfactors that can attenuate the TCA cycle, and (ii) improving PDHC activity in the ΔramAstrain.
Table 1: Fermentation profiles of the C. glutamicum strains analyzed. The data are meanvalues and standard deviation from three independent experiments. For growth andmetabolites, the values represent the maxima.
Parameter

Wild typeΔramA

Δ4Δ4 ΔramA

Specific growth rate (h-1)a0.470 ± 0.009

0.443 ± 0.005c0.494 ± 0.011c

0.425 ± 0.009d

Growth (OD600)

27.4 ± 0.7

23.0 ± 0.2c

27.7 ± 0.5

20.7 ± 0.4d

Glucose consumption rateb

0.957 ± 0.045

1.27 ± 0.04c

0.913 ± 0.016c

1.22 ± 0.01d

Pyruvate (mM)

46.9 ± 3.8

44.2 ± 4.8

89.3 ± 4.0c

161 ± 4d

Lactate (mM)

43.8 ± 15.5

103 ± 10c

4.31 ± 0.26c

5.87 ± 0.12d

Acetate (mM)

76.9 ± 10.5

109 ± 4c13.5 ± 0.4c

12.7 ± 0.6

Succinate (mM)

18.5 ± 0.3

13.8 ± 0.2c

26.6 ± 2.5c

18.9 ± 0.7d

2-Oxoglutarate (mM)

5.26 ± 2.75

0.687 ± 0.203c

4.01 ± 0.03

2.97 ± 0.18d

a The specific growth rates were calculated from data obtained during the logarithmic growth phase(between 3 h and 6 h).
b

The glucose consumption rates were calculated from data obtained during the early stationary

phase (between 6 h and 15 h) and were expressed as consumed glucose (mM)·(averageOD600)-1·h-1.
c Statistically significant differences (t-test P-values < 0.05), when compared to the wild-type strain.
d Statistically significant differences (t-test P-values <0.05), when compared to the Δ4 strain.

2. Construction of pyruvate-producing mutants
In the context of the second approach proposed above (improving PDHC activity in theΔramA strain), using as host a strain that can accumulate high amount of pyruvate, aprecursor of acetyl-CoA, would be favorable. To date, a prototrophic pyruvate-producing
strain of C. glutamicum (i.e., a strain without inactivation of PDHC) has not been reported[3].
The experiments described in section 1. have shown that the deletion of ramA led to afermentative phenotype. Therefore, a mutant strain lacking the enzymes involved in all
known lactate- and acetate-producing pathways (i.e., LDH, PQO, CAT, and PTA/AK; seeFig. 1) was constructed, referred to as Δ4. The effects of ramA deletion on glucose
metabolism were then evaluated in this mutant (Table 1). The Δ4 strain produced about twotimes more pyruvate than the wild-type and ΔramA strains. Moreover, the pyruvate titerfurther increased in the Δ4 ΔramA strain and was almost two times higher than that in the Δ4strain. Furthermore, compared to the Δ4 strain, the Δ4 ΔramA strain showed an increased
glucose consumption rate, decreased growth, and lower accumulation of 2-oxoglutarate andsuccinate. These results indicate that the TCA cycle is attenuated in the Δ4 ΔramA strain, as
is the case in the ΔramA strain[5].
Conclusions
This study aimed to (i) identify factors that can increase the pool of acetyl-CoA and (ii)apply this knowledge to the production of acetyl-CoA-derived compounds in C. glutamicum.
At this stage of the project, I have made very encouraging progress towards the first aim. I
have shown that ramA deletion can effectively attenuate the TCA cycle, and I have generated
a C. glutamicum strain that can produce high levels of pyruvate, a precursor of acetyl-CoA.
RamA has been reported to positively regulate the expression of the aceF gene, which
encodes the E2 subunit of PDHC (formerly known as sucB and assumed to encode the E2
subunit of ODHC)[1,4], likely explaining the decreased PDHC activity observed in the ΔramA
strain and providing a clue for prospective work. Furthermore, the amino acid sequence ofAceF differs between C. glutamicum strains, and data suggest that these differences affect
PDHC activity. In the near future, I would like to further explore these lines of investigation
and achieve the objectives of this research project.
References
1) Auchter, M. et al. (2011) RamA and RamB are global transcriptional regulators inCorynebacterium glutamicum and control genes for enzymes of the central metabolism. J.
Biotechnol. 154: 126-139.
2) van Ooyen, J., Emer, D., Bussmann, M., Bott, M., Eikmanns, B.J., and Eggeling, L.
(2011) Citrate synthase in Corynebacterium glutamicum is encoded by two gltA
transcripts which are controlled by RamA, RamB, and GlxR. J. Biotechnol. 154:140-148.
3) Wieschalka, S., Blombach, B., and Eikmanns, B.J. (2012) Engineering Corynebacterium
glutamicum for the production of pyruvate. Appl. Microbiol. Biotechnol. 94: 449-459.
4) Hoffelder, M., Raasch, K., van Ooyen, J., and Eggeling, L. (2010) The E2 domain ofOdhA of Corynebacterium glutamicum has succinyltransferase activity dependent on
lipoyl residues of the acetyltransferase AceF. J. Bacteriol. 192: 5203-5211.
5) Kataoka, N., Vangnai, A.S., Pongtharangkul, T., Yakushi, T., Wada, M., Yokota, A.,
Matsushita, K. (2019) Engineering of Corynebacterium glutamicum as a prototrophic
pyruvate-producing strain: Characterization of a ramA-deficient mutant and its
application for metabolic engineering. Biosci. Biotechnol. Biochem. 83: 372-380.