Folmer Fredslund

Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Forming indigo directly in the yarn through indican (indoxyl-β-glucoside) is a promising alternative route with mild conditions. Indican eliminates the requirement for reducing agent while still ending as indigo, the only known molecule yielding the unique hue of blue denim. However, a bulk source of indican is missing. Here, we employ enzyme and process engineering guided by techno-economic analyses to develop an economically viable drop-in indican synthesis technology. Rational engineering of PtUGT1, a glycosyltransferase from the indigo plant, alleviated the severe substrate inactivation observed with the wildtype enzyme at the titers needed for bulk production. We further describe a mild, light-driven dyeing process. Finally, we conduct techno-economic, social sustainability, and comparative life-cycle assessments. These indicate that the presented technologies have the potential to significantly reduce environmental impacts from blue denim dyeing with only a modest cost increase.

The determined 3D structure of a fungal glucuronan lyase and mutational studies reveal that the lyases from PL7_4 subfamily employ a single tyrosine as both base and acid catalyst to degrade β-(1,4) glucuronan via a syn β-elimination mechanism.

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea ( Am SP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, Am SP-Q353F and Am SP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89% of a novel compound (+)-catechin-4′-O-α-D-glucopyranoside (CAT-4’) for AmSP-P140D and 92% of (+)-catechin-3′-O-α-D-glucopyranoside (CAT-3’) for AmSP-Q353F. The compound CAT-4’ was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: Am SP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4’ while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3’.

Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Several innovations aim to replace the damaging chemicals and processes with ecologically attractive alternatives, but the economic and social aspects of sustainability are often overlooked, resulting in poor implementation. Forming indigo directly in the yarn through indican (indoxyl-β-glucoside) is a promising route with mild conditions. However, a sustainable source of indican is missing. We employed enzyme and process engineering guided by techno-economic analyses to develop an economically viable drop-in indican synthesis technology. Engineering of PtUGT1, a glycosyltransferase from the indigo plant, alleviated the severe substrate inactivation observed at the titers needed for bulk production. We further discovered a mild, light-driven dyeing process. Techno-economic, social sustainability, and comparative life-cycle assessments showed that these novel technologies have the potential to significantly reduce dyeing impacts.

Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and k_cat were limited, prohibiting the application of the engineered catalyst. Here, we identified a naturally regioselective silibinin glucosyltransferase, UGT706F8, a family 1 glycosyltransferase from Zea mays. It selectively and efficiently (k_cat = 2.1 ± 0.1 s^–1; K_M = 36.9 ± 5.2 μM; TTN = 768 ± 22) catalyzes the quantitative synthesis of silibinin 7-O-β-d-glucoside. We solved the crystal structure of UGT706F8 and investigated the molecular determinants of regioselective silibinin glucosylation. UGT706F8 was the only regioselective enzyme among 18 glycosyltransferases found to be active on silibinin. We found the temperature optimum of UGT706F8 to be 34 °C and the pH optimum to be 7–8. Our results indicate that UGT706F8 is an efficient silibinin glycosyltransferase that enables biocatalytic production of silbinin 7-O-β-d-glucoside.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, revealed a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expanded the AA7-substrate range and unveiled a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses revealed unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly transferred electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuelled cellulose degradation by LPMOs without exogenous reductants. The expansion of the substrate range and the synergistic interplay with LPMOs highlight the functional diversity within AA7 and set the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

Covering: up to 2020 C-Glycosyltransferases are enzymes that catalyse the transfer of sugar molecules to carbon atoms in substituted aromatic rings of a variety of natural products. The resulting β-C-glycosidic bond is more stable in vivo than most O-glycosidic bonds, hence offering an attractive modulation of a variety of compounds with multiple biological activities. While C-glycosylated natural products have been known for centuries, our knowledge of corresponding C-glycosyltransferases is scarce. Here, we discuss commonalities and differences in the known C-glycosyltransferases, review attempts to leverage them as synthetic biocatalysts, and discuss current challenges and limitations in their research and application.