Catalyst development for biochemical cascade reactions often follows a “whole‐cell‐approach” in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two‐enzyme co‐expression in E. coli, for whole‐cell‐production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co‐expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg−1) and sucrose phosphorylase (122 U mg−1). Mono‐ and bicistronic co‐expression strategies controlling transcription, transcription‐translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole‐cell‐catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min−1 g−1 dry cells) and performance metrics (30 g L−1; ∼82% yield; 3.8 g L−1 h−1 overall productivity) promising for cellobiose production.

Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

Background Soluble cello-oligosaccharides (COS, β‐1,4‐D‐gluco‐oligosaccharides with degree of polymerization DP 2–6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS. Results Here, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7lacO promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space–time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion. Conclusions A whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.

Background Fatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts. Results We investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media. Conclusions Baker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.