Background Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. Results Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii , a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum . Conclusion The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures. Electronic supplementary material The online version of this article (10.1186/s13068-019-1386-y) contains supplementary material, which is available to authorized users.

C−C bond-forming reactions often require nucleophilic carbon species rarely compatible with aqueous reaction media, thus restricting their appearance in biocatalysis.Here we report the use of nitroalkanes as a structurally versatile class of nucleophilic substrates for C−C bond formation catalyzed by variants of the β-subunit of tryptophan synthase (TrpB). The enzymes accept a wide range of nitroalkanes to form noncanonical amino acids, where the nitro group can serve as a handle for further modification. Using nitroalkane nucleophiles greatly expands the scope of compounds made by TrpB variants and establishes nitroalkanes as a valuable substrate class for biocatalytic C−C bond formation.

We previously engineered the tryptophan synthase b-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form CC bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N-C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.

Supplementary Information Additional Materials and Methods Strains, Media, and Growth Conditions Caldicellulosiruptor bescii and Escherichia coli strains used in this study are listed in Table S1. All C. bescii strains were grown anaerobically at 65°C on solid or in liquid low osmolarity defined (LOD) medium (1), as previously described, with .5 % w/v D(+)-Cellobiose (Acros Organics, NJ, U.S.A., Code: 108465000, Lot: A0384025) as the sole carbon source, final pH 6.8, for routine growth and transformation experiments (2). For growth of uracil auxotrophic strains based off of JWCB029 (ΔpyrFA ldh::ISCbe4 Δcbe1 ΔcelA), the LOD medium contained 40 µM uracil. This concentration of uracil does not support growth of C. bescii as sole carbon source. E. coli strain DH5α was used for plasmid DNA construction and preparation using standard techniques as described (3). E. coli cells were cultured in LB broth supplemented with apramycin (50 μg/mL) and plasmid DNA was isolated using a Qiagen Mini-prep kit (Qiagen, Hilden, Germany). Chromosomal DNA from Caldicellulosiruptor strains was extracted using the Quick-gDNA™ MiniPrep (Zymo Research, Irvine, CA, U.S.A.) as previously described (4). Construction and Transformation of CelA and CelA Derivative Expression Vectors Plasmids in this study were generated using Q5 High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA, U.S.A.), restriction enzymes (New England BioLabs, Ipswich, MA, U.S.A.), and Fast-link TM DNA Ligase (Epicentre Technologies, Madison, WI, U.S.A.) according to the manufacturer's instructions. For the construction of pJYW008 (Figure S1, Table S1), a 10.64 kb DNA fragment was synthesized with the reverse primer JY017 and forward primer JY018 using pDCW173 (4) as a template. After amplification, the PCR product was ligated via blunt-end ligation. The 9.96 kb (amplified using DCB151 and DCB152) and 10.62 kb (amplified using DCB152 and DCB153) PCR amplified DNA fragments, using pDCW173 as a template, were synthesized for the construction of pDCYB037 and pDCYB038 (Table S1), respectively. These two linear DNA fragments were digested with SphI and ligated to construct pDCYB037 and pDCYB038, respectively. The C. bescii CelA gene sequence (Cbes_1867; GenBank accession number Z86105) was codon optimized for expression in E. coli and cloned into a pET28b(+) vector using NcoI and XhoI sites (GenScript, Piscataway, NJ, U.S.A.). The sequence for a 6x histidine tag was placed at the C-terminus to facilitate protein purification. It was referred as pDCYB017 (Table S1). For the construction of pDCYB018 (Table S1), a 7.81 kb DNA fragment was synthesized with the reverse primer DCB068 and forward primer DCB069 using pDCYB017 as a template. After amplification, the PCR product was ligated via blunt-end ligation. The 7.78 kb (amplified using DCB155 and DCB157) and 7.11 kb (amplified using DCB157 and DCB256) PCR amplified DNA fragments, using pDCYB017 as a template, were synthesized for the construction of pDCYB075 and pDCYB076 (Table S1), respectively. These two linear DNA fra...