Katharina Schmölzer

Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

BackgroundEnzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(P)H. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH), presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S)-1-(2-chlorophenyl)ethanol from o-chloroacetophenone.ResultsAs compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by just 19% (1140 U/gCDW). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/gCDW) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.ConclusionsExpression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.

Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development

Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions.

Bioprocess design guided by in situ substrate supply and product removal: Process intensification for synthesis of (S)-1-(2-chlorophenyl)ethanol

Highlights ► o-Chloroacetophenone and its reduction product are highly toxic to enzymes and cells. ► Properties of substrate and product guided comprehensive bioprocess engineering. ► Substrate supply and product removal were optimized. ► Mass transfer limitations were overcome by cell permeabilization. ► (S)-1-(2-chlorophenyl)ethanol was obtained in high optical purity and yield.

Mechanistic study of CMP‐Neu5Ac hydrolysis by α2,3‐sialyltransferase from Pasteurella dagmatis

Bacterial sialyltransferases of the glycosyltransferase family GT‐80 exhibit pronounced hydrolase activity toward CMP‐activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP‐Neu5Ac by Pasteurella dagmatis α2,3‐sialyltransferase (PdST) occurs with axial‐to‐equatorial inversion of the configuration at the anomeric center to release the α‐Neu5Ac product. We propose a catalytic reaction through a single displacement‐like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His284 in the active site replaced by Asn, Asp or Tyr showed up to 104‐fold reduced activity, but catalyzed CMP‐Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His284 in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities

Backgroundα-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This “capping” of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins.ResultsHuman α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates.ConclusionsFunctional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 – 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Glycosyltransferase cascades made fit for chemical production: Integrated biocatalytic process for the natural polyphenol C-glucoside nothofagin

Glycosyltransferase cascades are promising tools of biocatalysis for natural product glycosylation, but their suitability for actual production remains to be shown. Here, we demonstrate at a scale of 100 g isolated product the integrated biocatalytic production of nothofagin, the natural 3′‐C‐β‐D‐glucoside of the polyphenol phloretin. A parallel reaction cascade involving coupled C‐glucosyltransferase and sucrose synthase was optimized for the one‐pot glucosylation of phloretin from sucrose via an UDP/UDP‐glucose shuttle. Inclusion complexation with the highly water soluble 2‐hydroxypropyl‐β‐cyclodextrin pushed the phloretin solubility to its upper practical limit (∼120 mM) and so removed the main bottleneck on an efficient synthesis of nothofagin. The biotransformation thus intensified had excellent performance metrics of 97% yield and ∼50 gproduct/L at a space‐time yield of 3 g/L/hr. The UDP‐glucose was regenerated up to ∼220 times. A scalable downstream process for efficient recovery of nothofagin (≥95% purity; ≥65% yield) was developed. A tailored anion‐exchange chromatography at pH 8.5 was used for capture and initial purification of the product. Recycling of the 2‐hydroxypropyl‐β‐cyclodextrin would also be possible at this step. Product precipitation at a lowered pH of 6.0 and re‐dissolution in acetone effectively replaced desalting by size exclusion chromatography in the final step of nothofagin purification. This study therefore, reveals the potential for process intensification in the glycosylation of polyphenol acceptors by glycosyltransferase cascades. It demonstrates that, with up‐ and downstream processing carefully optimized and suitably interconnected, a powerful biocatalytic technology becomes available for the production of an important class of glycosides difficult to prepare otherwise.

Combining expression and process engineering for high-quality production of human sialyltransferase in Pichia pastoris.

The human β-galactoside α2,6-sialyltransferase I, ST6Gal-I has drawn considerable interest for its use as biocatalyst for in-vitro glycoengineering of recombinantly produced therapeutic proteins. By attaching sialic acid onto the terminal galactoses of biantennary protein N-glycans, ST6Gal-I facilitates protein remodeling towards a humanized glycosylation and thus optimized efficacy in pharmacological use. Secreted expression of ST6Gal-I in Pichia pastoris is promising, but proteolysis restricts both the yield and the quality of the enzyme produced. Focusing on an N-terminally truncated (Δ108) variant of ST6Gal-I previously shown to represent a minimally sized, still active form of ST6Gal-I, we show here that protein expression engineering and optimization of bioreactor cultivation of P. pastoris KM71H (pPICZαB) synergized to enhance the maximum enzyme titer about 57-fold to 17units/L. N-Terminal fusion to the Flag-tag plus deletion of a potential proteolytic site (Lys(114)-Asn→Gln(114)-Asn) improved the intrinsic resistance of Δ108ST6Gal-I to degradation in P. pastoris culture. A mixed glycerol/methanol feeding protocol for P. pastoris growth and induction was key for enzyme production in high yield and quality. The sialyltransferase was recovered from the bioreactor culture in a yield of 70% using a single step of anion-exchange chromatography. Its specific activity was 0.05units/mg protein.

All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity

Sialyltransferases are important enzymes of glycobiology and the related biotechnologies. The development of sialyltransferases calls for access to quick, inexpensive, and robust analytical tools. We have established an assay for simultaneous characterization of sialyltransferase activity, error hydrolysis, and site selectivity. The described assay does not require expensive substrates, is very sensitive (limit of detection=0.3 μU), and is easy to perform. It is based on sialylation of nitrophenyl galactosides; the products thereof are separated and quantified by ion pair reversed phase high-performance liquid chromatography with ultraviolet detection.

Active‐site His85 of Pasteurella dagmatis sialyltransferase facilitates productive sialyl transfer and so prevents futile hydrolysis of CMP‐Neu5Ac

Sialyltransferases of the GT‐80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans‐sialylation, and hydrolysis activities. How these enzymes utilize their active‐site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild‐type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3‐ but also the α2,6‐sialylated product (21 %). The H85N variant was virtually inactive in trans‐sialylation but showed almost the same CMP‐Neu5Ac hydrolase activity as wild‐type. The competition between sialyl transfer and hydrolysis in the conversion of CMP‐Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m−1 for the H85N variant, compared to 17 000 m−1 for the wild‐type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP‐Neu5Ac in the reaction.