Green Chemistry
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where it was shown that the tailored variants had a >30 times
lower Km value and significantly optimized catalytic efficiency
compared to the wild type.
The two most promising variants (FLS_A3 and FLS_B2)
were combined in a cell-free synthetic cascade and the pro-
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1
duction of ERY from the C carbon source was shown for the
first time. In this setting, the variant catalyzing FALD fusion to
GALD was identified as a bottleneck in achieving complete
conversion of FALD; thus, the catalytic activity of FLS_A3 must
be further optimized. As the primary objective of the in silico
investigation being ERY formation from GALD (second reac-
tion), it was more plausible that highly suitable variants
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(FLS_B1 and FLS_B2) were found for this reaction.
The potential of these variants was demonstrated in com-
parison to the wild type in a biocatalytic process starting from
2
5.0 g L− GALD, which was recently described as a promising
1
5 S. Desmons, R. Fauré and S. Bontemps, ACS Catal., 2019, 9,
9575–9588.
2
7,28
renewable C platform molecule.
FLS_B2 allowed the pro-
2
duction of 24.6 g L− ERY with a theoretical yield of 98%. This
represents the highest ERY concentration achieved in vitro
reported to date. Furthermore, we were able to show that this
biocatalyst does not undergo significant degradation or confor-
mational change during the biocatalysis. This additionally
highlights the robustness of the FLS.
1
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J. B. Siegel, A. L. Smith, S. Poust, A. J. Wargacki, A. Bar-
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E. Noor, J. L. Gallaher, J. Bale, Y. Yoshikuni, M. H. Gelb,
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Author contributions
S.G., A.P. and V.S. conceived and designed the study. S.G. per-
formed in silico studies, preparation of the library, screening
development, screening, selection of variants, characterization,
applying suitable variants, determination of kinetic para-
meters and data analysis. V.W. prepared library supernatant
and performed GDH assay. S.G. wrote the initial draft of the
9 S. Poust, J. Piety, A. Bar-Even, C. Louw, D. Baker,
J. D. Keasling and J. B. Siegel, ChemBioChem, 2015, 16,
1950–1954.
manuscript. V.W., A.P. and V.S. reviewed the manuscript. All 10 S. Eymur, Turk. J. Chem., 2013, 37(4), 586–609.
authors read and approved the final version of the manuscript. 11 (a) L. Zhang, R. Singh, S. D. Z. Guo, J. Li, F. Chen, Y. He,
X. Guan, Y. C. Kang and J.-K. Lee, Green Chem., 2018, 20,
230–242; (b) T. Li, Z. Tang, H. Wei, Z. Tan, P. Liu, J. Li,
Y. Zheng, J. Lin, W. Liu, H. Jiang, H. Liu, L. Zhu and Y. Ma,
Green Chem., 2020, 22, 6809–6814.
Conflicts of interest
André Pick is the CEO of CASCAT GmbH, a biotechnology 12 J. Yang, S. Sun, Y. Men, Y. Zeng, Y. Zhu, Y. Sun and Y. Ma,
company pursuing commercialization of bio-based processes.
Catal. Sci. Technol., 2017, 7, 3459–3463.
1
1
3 J. E. Hodge, J. Agric. Food Chem., 1953, 1, 928–943.
4 R. Jermann, M. Toumiat and D. Imfeld, Int. J. Cosmet. Sci.,
2
002, 24, 35–42.
Acknowledgements
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5 T. Schreier and R. Jermann, Combination of erythrulose
and a reducing sugar with self-tanning properties, Google
Patents, 2002.
Fruitful discussions with Samuel Sutiono and Ioannis Zachos,
as well as invaluable feedback and proofreading the manu-
script are greatly appreciated. Samed Güner thanks Dr Stefan 16 C. Burger, C. Kessler, S. Gruber, A. Ehrenreich, W. Liebl
Gilch (Evonik Industries AG) for his excellent mentoring.
and D. Weuster-Botz, Appl. Microbiol. Biotechnol., 2019, 103,
393–4404.
4
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7 F. Carly and P. Fickers, Yeast, 2018, 35, 455–463.
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