Generalizability of Soft Sensors for Bioprocesses through Similarity Analysis and Phase-Dependent Recalibration.

A soft sensor concept is typically developed and calibrated for individual bioprocesses in a time-consuming manual procedure. Following that, the prediction performance of these soft sensors degrades over time, due to changes in raw materials, biological variability, and modified process strategies. Through automatic adaptation and recalibration, adaptive soft sensor concepts have the potential to generalize soft sensor principles and make them applicable across bioprocesses. In this study, a new generalized adaptation algorithm for soft sensors is developed to provide phase-dependent recalibration of soft sensors based on multiway principal component analysis, a similarity analysis, and robust, generalist phase detection in multiphase bioprocesses. This generalist soft sensor concept was evaluated in two multiphase bioprocesses with various target values, media, and microorganisms. Consequently, the soft sensor concept was tested for biomass prediction in a Pichia pastoris process, and biomass and protein prediction in a Bacillus subtilis process, where the process characteristics (cultivation media and cultivation strategy) were varied. High prediction performance was demonstrated for P. pastoris processes (relative error = 6.9%) as well as B. subtilis processes in two different media during batch and fed-batch phases (relative errors in optimized high-performance medium: biomass prediction = 12.2%, protein prediction = 7.2%; relative errors in standard medium: biomass prediction = 12.8%, protein prediction = 8.8%).

Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

BACKGROUND: The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium Clostridium thermocellum is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass. RESULTS: In this study, 60 cellulosomal components were recombinantly produced in E. coli and systematically permuted in synthetic complexes to study the function-activity relationship of all available enzymes on Kraft pulp from pine wood as the substrate. Starting from a basic exo/endoglucanase complex, we were able to identify additional functional classes such as mannanase and xylanase for optimal activity on the substrate. Based on these results, we predicted a synthetic cellulosome complex consisting of seven single components (including the scaffoldin protein and a ß-glucosidase) and characterized it biochemically. We obtained a highly thermostable complex with optimal activity around 60-65 °C and an optimal pH in agreement with the optimum of the native cellulosome (pH 5.8). Remarkably, a fully synthetic complex containing 47 single cellulosomal components showed comparable activity with a commercially available fungal enzyme cocktail on the softwood pulp substrate. CONCLUSIONS: Our results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of C. thermocellum can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.

Downstream processing of serinol from a glycerol-based fermentation broth and transfer to other amine containing molecules.

A possible application of glycerol, which is produced in large amounts as a by-product from the biodiesel industry, is its fermentation to serinol (2-amino-1,3-propanediol), a glycerol derivative. The downstream processing of this glycerol-based fermentation broth was investigated. The challenge of the isolation of serinol was the complex media and the solubility of the desired substance in aqueous media. In this study, the isolation of serinol was investigated by an appropriate reversible derivatization method. Serinol was isolated by protecting the amino group with diethyl ethoxymethylenemalonate directly in the aqueous phase, followed by extraction of the 2,2-bis(ethoxycarbonyl)vinyl-serinol (BECV-serinol) with ethyl acetate resulting in an isolated yield of 63%. We demonstrate the possibility of isolation of a hydroscopic amino alcohol from the fermentation broth and the comparison of the products in water as well as the cleavage of the 2,2-bis(ethoxycarbonyl)vinyl group (BECV group). The procedure can also be used for other amino group containing molecules, such as serine, glucosamine, hexylamine and amino methyl laureate.

Strain and process development for poly(3HB-co-3HP) fermentation by engineered Shimwellia blattae from glycerol.

Poly(3-hydroxybytyrate-co-3-hydroxypropionate), poly(3HB-co-3HP), is a possible alternative to synthetic polymers such as polypropylene, polystyrene and polyethylene due to its low crystallinity and fragility. We already reported that recombinant strains of Shimwellia blattae expressing 1,3-propanediol dehydrogenase DhaT as well as aldehyde dehydrogenase AldD of Pseudomonas putida KT2442, propionate-CoA transferase Pct of Clostridium propionicum X2 and PHA synthase PhaC1 of Ralstonia eutropha H16 are able to accumulate up to 14.5% (wtPHA/wtCDW) of poly(3-hydroxypropionate), poly(3HP), homopolymer from glycerol as a sole carbon source (Appl Microbiol Biotechnol 98:7409-7422, 2014a). However, the cell density was rather low. In this study, we optimized the medium aiming at a more efficient PHA synthesis, and we engineered a S. blattae strain accumulating poly(3HB-co-3HP) with varying contents of the constituent 3-hydroxypropionate (3HP) depending on the cultivation conditions. Consequently, 7.12, 0.77 and 0.32 gPHA/L of poly(3HB-co-3HP) containing 2.1, 8.3 and 18.1 mol% 3HP under anaerobic/aerobic (the first 24 hours under anaerobic condition, thereafter, aerobic condition), low aeration/agitation (the minimum stirring rate required in medium mixing and small amount of aeration) and anaerobic conditions (the minimum stirring rate required in medium mixing without aeration), respectively, were synthesized from glycerol by the genetically modified S. blattae ATCC33430 strains in optimized culture medium.

Poly(3-hydroxypropionate): a promising alternative to fossil fuel-based materials.

Polyhydroxyalkanoates (PHAs) are storage compounds synthesized by numerous microorganisms and have attracted the interest of industry since they are biobased and biodegradable alternatives to fossil fuel-derived plastics. Among PHAs, poly(3-hydroxypropionate) [poly(3HP)] has outstanding material characteristics and exhibits a large variety of applications. As it is not brittle like, e.g., the best-studied PHA, poly(3-hydroxybutyrate) [poly(3HB)], it can be used as a plasticizer in blends to improve their properties. Furthermore, 3-hydroxypropionic acid (3HP) is considered likely to become one of the new industrial building blocks, and it can be obtained from poly(3HP) by simple hydrolysis. Unfortunately, no natural organism is known to accumulate poly(3HP) so far. Thus, several efforts have been made to engineer genetically modified organisms capable of synthesizing the homopolymer or copolymers containing 3HP. In this review, the achievements made so far in efforts to obtain biomass which has accumulated poly(3HP) or 3HP-containing copolymers, as well as the properties of these polyesters and their applications, are compiled and evaluated.

Influence of the operon structure on poly(3-hydroxypropionate) synthesis in Shimwellia blattae.

Glycerol has become a cheap and abundant carbon source due to biodiesel production at a large scale, and it is available for several biotechnological applications. We recently established poly(3-hydroxypropionate) [poly(3HP)] synthesis in a recombinant Shimwellia blattae strain (Heinrich et al. Appl Environ Microbiol 79:3582-3589, 2013). The major drawbacks of the current strains are (i) low poly(3HP) yields, (ii) low plasmid stability and (iii) insufficient conversion rates. In this study, we demonstrated the influence of alterations of the operon structure, consisting of 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate:coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2 and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16. It was shown that S. blattae ATCC33430/pBBR1MCS-2::dhaT::pct::aldD::phaC1 synthesized up to 14.5 % (wtPHA/wtCDW) in a 2-L fed-batch fermentation process. Furthermore, we overcame the problem of plasmid losses during the fermentation period by engineering a carbon source-dependent plasmid addiction system in a triose phosphate isomerase knockout mutant. An assumed poly(3-hydroxyalkanoic acid) degrading activity of the lipase/esterase YbfF could not be confirmed.

From waste to plastic: synthesis of poly(3-hydroxypropionate) in Shimwellia blattae.

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.

Biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol) in recombinant Escherichia coli.

Microbial conversion is an important technology for the refinement of renewable resources. Here, we describe the biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol), a relevant intermediate in several chemical syntheses processes. Either the dihydroxyacetone phosphate aminotransferase/dihydrorhizobitoxine synthase (RtxA) of Bradyrhizobium elkanii USD94 or only the N-terminal domain (RtxA513) comprising the first reaction, respectively, was expressed in recombinant Escherichia coli. Serinol contents of up to 3.3 g/l were achieved in batch cultures. We could further clarify that glutamic acid is the preferred cosubstrate for the transamination of dihydroxyacetone phosphate to serinolphosphate, which is the essential step in serinol synthesis. An in vivo detoxification of serinol employing wax ester synthase/acyl-CoA:diacyl-glycerol acyl transferase from Acinetobacter baylyi ADP1 was not accomplished. This study paves the way for biotechnological production of serinol from glycerol derived from the biodiesel industry.

Serinol: small molecule - big impact.

The amino alcohol serinol (2-amino-1,3-propanediol) has become a common intermediate for several chemical processes. Since the 1940s serinol was used as precursor for synthesis of synthetic antibiotics (chloramphenicol). In the last years, new scopes of applications were discovered. Serinol is used for X-ray contrast agents, pharmaceuticals or for chemical sphingosine/ceramide synthesis. It can either be obtained by chemical processes based on 2-nitro-1,3-propanediol, dihydroxyacetone and ammonia, dihydroxyacetone oxime or 5-amino-1,3-dioxane, or biotechnological application of amino alcohol dehydrogenases (AMDH) or transaminases. This review provides a survey of synthesis, properties and applications for serinol.

Biosynthesis and biodegradation of 3-hydroxypropionate-containing polyesters.

3-Hydroxypropionate (3HP) is an important compound in the chemical industry, and the polymerized 3HP can be used as a bioplastic. In this review, we focus on polyesters consisting of 3HP monomers, including the homopolyester poly(3-hydroxypropionate) and copolyesters poly(3-hydroxybutyrate-co-3-hydroxypropionate), poly(3-hydroxypropionate-co-3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate), and poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-4-hydroxybutyrate-co-lactate). Homopolyesters like poly(3-hydroxybutyrate) are often highly crystalline and brittle, which limits some of their applications. The incorporation of 3HP monomers reduces the glass transition temperature, the crystallinity, and also, at up to 60 to 70 mol% 3HP, the melting point of the copolymer. This review provides a survey of the synthesis and physical properties of different polyesters containing 3HP.

Conversion of glycerol to poly(3-hydroxypropionate) in recombinant Escherichia coli.

We have developed the conversion of glycerol into thermoplastic poly(3-hydroxypropionate) [poly(3HP)]. For this, the genes for glycerol dehydratase (dhaB1) of Clostridium butyricum, propionaldehyde dehydrogenase (pduP) of Salmonella enterica serovar Typhimurium LT2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha were expressed in recombinant Escherichia coli. Poly(3HP) was accumulated up to 11.98% (wt/wt [cell dry weight]) in a two-step, fed-batch fermentation. The present study shows an interesting application to engineer a poly(3HP) synthesis pathway in bacteria.