Development of an Escherichia coli-based whole-cell catalyst
expressing metalloenzymes capable of anaerobic CO2 fixation
Takashi FUJISHIRO
Department of Biochemistry and Molecular Biology, School of Science,
Saitama University
Report: Genes encoding several types of anaerobic metalloenzymes involved in CO2
metabolism were cloned and incorporated into Escherichia coli cells to create various E. colitransformants. We confirmed that these transformed E. coli cells can grow like wild-type
cells under aerobic conditions, indicating that the transformants can be utilized in anaerobic
growth experiments. Based on the results, screening of anaerobic growth conditions for CO2
metabolism was tested using the E. coli transformants.
Research aims
CO2 has been extensively studied in a wide range of research areas because CO2 is agreenhouse gas that contributes to global warming and is a source for carbon recycling. Thus,
development of a system for catalyzing atmospheric CO2 fixation is of great interest. In
particular, the creation of whole-cell catalysts by the "synthetic biology" strategy of
introducing several genes responsible for desired metabolism into a model organism (e.g.
Escherichia coli) has been extensively pursued. Whole-cell catalysts are attractive
biocatalytic systems that can offer the following benefits: (1) some enzymes that are unstablein vitro can be used without loss of activity in cellular environments; and (2) some cofactors
required for the targeted enzymes are supplied by the biosynthetic systems in the host cells.
In this study, genes encoding the metalloenzymes, CO dehydrogenase, acetyl-CoA synthase,and hydrogenase, which are involved in anaerobic CO2 metabolism, were introduced into E.
coli cells for creating an anaerobic CO2-metabolizing whole-cell biocatalyst. In this E.
coli-based whole-cell catalytic system, reducing power is supplied by hydrogenase activity,with H2 supplied as a substrate. The rest of the CO2 reduction system is composed of CO
dehydrogenase, and CO-utilization by acetyl-CoA synthase yields acetyl-CoA. The resulting
E. coli transformant is regarded as an artificial whole-cell catalyst of anaerobic CO2-fixation.
At the beginning of this study, CO dehydrogenase, a CO2-CO interconverting enzyme, andhydrogenase, a hydrogen-activating enzyme, were tested for screening. We also note that CO
dehydrogenase and hydrogenase are iron-sulfur (Fe-S) cluster-containing enzymes, and theendogenous quantity of Fe-S clusters in E. coli is not expected to be enough to supply theover-expressed CO dehydrogenase and hydrogenase. However, our research group has been
investigating an Fe-S cluster biosynthetic gene cluster, the so-called isc operon, which can beused to overexpress Fe-S proteins in E. coli (Fe-S cluster-rich E. coli).3 ) Other Fe-S cluster
biosynthetic gene clusters, e.g., suf and suf-like operons, are also known. In general, the Fe-Scluster-binding efficiency of overexpressed Fe-S proteins is dependent on the choice of Fe-Scluster biosynthetic genes; hence, the Fe-S cluster biosynthetic operons should also be
optimized by screening, together with the CO dehydrogenase- and hydrogenase-encoding
genes.
Methods
Two types of CO dehydrogenases: 1) The Desulfovibrio vulgaris cooS gene encoding abacterial CO dehydrogenase and the Methanosarcina barkeri cooSαε gene encoding anarchaeal CO dehydrogenase were cloned into E. coli expression vectors, yielding
pACYC-cooS and pCDF-cooSαε vectors, respectively. For hydrogenase expression,2) wecloned two types of hydrogenase genes: the hyaAB genes (a catalytic domain of E. coli
[NiFe]-hydrogenase) derived from the E. coli hyaABCDEF operon, and the Clostridium
pasteurianum hydA gene encoding an [FeFe]-hydrogenase, which is not present in the E. coligenome, resulting in pRSF-hyaA and pET21d-hydA vectors. In addition to hydA,[FeFe]-hydrogenase maturation genes, hydE, hydF, and hydG, were also cloned, to yield
pACYC-hydE-hydF and pRSF-hydG vectors. These vectors were introduced into E. coli
C41(DE3) cells, to make CooS+HyaAB, CooSαε+HyaAB, and CooSαε+HydAEFG systems.
An artificial CooSαε-HyaAB fusion protein was also designed and constructed because afused protein of CooSαε and HyaAB was expected to facilitate intra-molecular electrontransfer to accelerate catalysis. We screened three candidate Fe-S cluster biosynthetic gene
operons: isc, suf, and suf-like operons. For isc, the traditionally used pRK-isc expression
vector and the newly created pBBR5-isc vector were tested for Fe-S cluster production in E.
coli cells. The suf and suf-like operons were cloned into the pRK vector and/or the pBBR5vector, followed by testing for Fe-S cluster production as with the isc operon.
Results
The E. coli C41(DE3) cells transformed with the expression vectors for the CO
dehydrogenase and hydrogenase genes were cultivated aerobically with isopropyl
β-D-1-thiogalactopyranoside (IPTG), and then, supernatants were analyzed by SDS-PAGE.
All introduced CO dehydrogenase and hydrogenase proteins were found to express in thesecells. However, their expression levels were different: D. vulgaris CooS, HydA, HydE, HydF,and HydG were well-expressed; M. barkeri CooSαε, HyaAB, and CooSαε-fusion-HyaAB
were weakly expressed. Among the Fe-S cluster biosynthetic operons, pBBR5-isc-

transformed E. coli cells displayed a higher yield of Fe-S clusters than pRK-isc-transformed
cells, judged by the brown color of the cells (Fe-S clusters are brown). The pRK-suf,pBBR5-suf, and pRK-suf-like vectors were also tested but yielded worse Fe-S cluster
production in E. coli than pBBR5-isc. Unfortunately, a pBBR5-suf-like vector could not beconstructed for these studies. These construction efforts are ongoing. We note that thecloning of each component gene in the suf-like operon was successful, and some proteinsencoded by suf-like genes have been overexpressed and purified for use in biochemical
assays.4) Therefore, one approach to cloning the suf-like operon into the pBBR5 vector
would be to use the Gibson assembly technique, where each of the genes in the suf-likeoperon is amplified and assembled in vitro into the pBBR5 vector. Next, these plasmids
would be incorporated into pBBR-isc-transformed E. coli C41(DE3) that would becultivated aerobically. If they grow aerobically at the same rate as wild-type cells, theywould next be applied to anaerobic cultivations with different ingredients/supplements toassess CO2 fixation in future works.
Conclusion
CO dehydrogenase genes (cooS and cooSαε) and hydrogenase genes (hyaAB andhydAEFG) were incorporated into E. coli cells harboring pBBR5-isc (Fe-S cluster-rich E.
coli cells). The created cells grew under aerobic conditions at the same levels as wild-type.
For future work, the created cells will be tested under anaerobic conditions for growth on
different types of carbon and hydrogen sources as anaerobic bacterial/archaeal metabolisms.
References
1 ） Adam, P. S., Borrel, G., Gribaldo, S. (2018) Evolutionary history of carbon monoxide
dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes. Proc. Natl. Acad.
Sci. USA 115: E1166-E1173.
2 ） Vignais, P. M., Billoud, B. (2007) Occurrence, classification, and biological function ofhydrogenases: an overview. Chem. Rev. 107: 4206-4272.
3）M. Nakamura, Y. Takahashi. (1999) Hyperproduction of recombinant ferredoxins in Escherichia
coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster. J.
Biochem. 126: 10-18.
4) R. Nakamura, M. Hikita, S. Ogawa, Y. Takahashi, T. Fujishiro. (2019) Snapshots ofPLP-substrate and PLP-product external aldimines as intermediates in two types ofcysteine desulfurase enzymes, FEBS J. accepted.