Development of a hybrid bio-conversion system consisting of invivo fermentation and in vitro enzymatic conversion
Kohsuke HONDA
International Center for Biotechnology, Osaka University
Research objective
The biocatalytic manufacturing of chemical compounds can be divided into two groups. The
first is fermentation, in which the metabolic activity of living microorganisms is used, and thesecond is enzymatic conversion, which employs only specific enzyme(s) isolated from living
cells as catalytic elements. Fermentation is preferred for the conversion of cheap agriculturalmaterials (e.g., starch and glucose) into primary metabolites, such as alcohols, organic acids,and amino acids, and is frequently implemented on a large scale. However, a certain fractionof the substrate is consumed for cell proliferation and maintenance; therefore, the product yield
is not always high. Meanwhile, enzymatic conversion is applicable even to chemicallysynthesized substrates, and is also able to achieve a high product yield owing to the absenceof unwanted side reactions. However, it often requires costly and time-consuming proceduresfor the isolation and purification of enzyme(s) from living cells.
In the present study, we aimed to develop a new biocatalytic chemical manufacturing systemthat has the advantages of both fermentation and enzymatic conversion. In the new system,
we can switch the process from the fermentation step (at 30–40℃) to the enzymatic conversion
step (at 50–70℃) by simply shifting the reaction temperature. Enzymes involved in the later
step are obtained from thermophilic microorganisms, andtheir genes are expressed in mesophilic microorganisms,
such as Escherichia coli and Corynebacterium glutamicum,
which are capable of fermentative production of a primarymetabolite. By shifting the reaction temperature, hostderived mesophilic enzymes are thermally inactivated, andonly recombinantly-produced thermophilic enzymes
remain in active forms. Consequently, the fermentatively
produced metabolite can be converted into a target productat a high yield (Figure 1). In the present study, wedemonstrated the one-pot production of L-pipecolic acid(L-PA) via fermentative production of L-lysine fromglucose to prove the concept of this hybrid system (Figure2).

Figure 1 Outline of thehybrid bio-conversion systemdeveloped in this study.

Figure 2 Enzymatic conversion of L-lysine to L-PAMethods
Homology searches were performed against the genome sequences of thermophiles stored in
our laboratory to explore thermophilic genes encoding enzymes involved in the conversion ofL-lysine to L-PA. Genes encoding lysine 6-dehydrogenase (Lys6DH) of Geobacillus
stearothermophilus (1) and pyrroline-5-carboxylate reductase (ProC) of Sulfolobus
solfataricus (2) were used as query sequences. Selected genes were expressed in E. coli, andthe resulting recombinant proteins were semi-purified by heat treatment of the cell extract.
The heat-purified enzymes were assayed, and the one that exhibited the highest specificactivities was used for further study. Genes encoding these enzymes were codon-optimized
for expression in C. glutamicum and integrated into a gene expression plasmid vector,
pECXT99A. The vector was then introduced into a metabolically engineered L-lysineproducing strain, C. glutamicum GRLys1 (3). The resulting transformant was aerobicallycultivated at 30℃ in 50 mL of CGXII medium supplemented with 4% glucose. Afterconfirmation of glucose depletion in the culture medium, the cultivation temperature wasincreased to 50℃ and NADH was added to the medium at a final concentration of 1 mM. LLysine and L-PA in the culture medium were determined by HPLC after derivatization withphenyl isothiocyanate.
Results
A homology search revealed that numerous thermophiles possess genes encoding putative
Lys6DH and ProC in their genomes. Among them, five genes were selected for Lys6DH andProC, and recombinant enzymes were prepared and assayed. Consequently, Lys6DH derived
from Geobacillus sp. 30 and ProC from Desulfurobacterium thermolithotrophum wereselected owing to their high specific activities among the tested enzymes. Genes encoding
these enzymes were tandemly aligned, introduced into an expression vector, and thenexpressed in C. glutamicum GRLys1. When the resulting transformant was cultivated at 30℃
in the CGXII medium, 39 mM L-lysine was accumulated in the medium after cultivation for
48 h. Meanwhile, no detectable amount of L-PA was found in the medium. A fraction of thecells was harvested from the culture medium and subjected to enzyme assay at 50℃. Theassay showed that both Lys6DH and ProC activities were detectable in the crude extract of C.
glutamicum GRLys1. This indicates that genes encoding thermophilic Lys6DH and ProC werefunctionally expressed in C. glutamicum but could not exert their catalytic activity at a reaction
temperature of 30℃. The cultivation temperature was then increased to 50℃, and NADH wasadded to the mixture at a final concentration of 1 mM. The mixture was further incubated for
48 h, resulting in the accumulation of 13 mM L-PA.
Conclusion
In the present study, fermentative production of L-lysine by metabolically engineered C.
glutamicum followed by the enzymatic conversion of the resulting L-lysine to L-PA wasachieved in a one-pot reaction. However, the conversion yield of L-PA from L-lysine in the
enzymatic conversion step remained moderate (33 mol%). Further studies will be performed
to address this issue, for example, by enhancing thermophilic enzyme gene expression levels
using engineered promoters and ribosome binding sites, and by increasing the cellconcentration (and thereby enzyme concentration) by employing a fed-batch operation in thefermentation step.
References
1) Heydari, M., Ohshima, T., Nunoura-Kominato, N., and Sakuraba, H. (2004) Highly stable
L-lysine dehydrogenase from the thermophile Geobacillus stearothermophilus isolated
from a Japanese hot spring: Characterization, gene cloning and sequencing, andexpression. Appl. Environ. Microbiol. 70: 937-942.
2) Meng, Z., Liu, Z., Lou, Z., Gong, X., Cao, Y., Bartlam, M., Zhang, K., Rao, Z. (2009)
Purification, characterization and crystallization of pyrroline-5-carboxylatereductase
from the hyperthermophilic archeon Sulfolobus solfataricus. Protein Expr. Purif. 64: 125130.
3) Pérez-García, F., Peters-Wendisch, P., and Wendisch, V. F. (2016) Engineering
Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid. Appl.
Microbiol. Biotechnol. 100: 8075-8090.
Acknowledgement
We thank Dr. Volker Wendish and Ms. Anastasia Kerbs (Bielefeld University) for kindly
donating C. glutamicum GRLys1, and for their valuable suggestions for this study.