Biocatalysis with in-situ product removal improves p-coumaric acid production.

Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10% salt solution was found to reduce tyrosine ammonia lyase activity by 19%, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40°C and pH 10 were found to be 3.8% salt and 9.5% polymer, and a 5% potassium phosphate and 12.5% poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for in situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33 g/L p-coumaric acid within 24 hours, a 1.9-fold improvement compared to biocatalysis without in situ product removal.

Raman spectroscopy and one-dimensional convolutional neural network modeling as a real-time monitoring tool for in vitro transaminase-catalyzed synthesis of a pharmaceutically relevant amine precursor.

Raman spectroscopy has been used to measure the concentration of a pharmaceutically relevant model amine intermediate for positive allosteric modulators of nicotinic acetylcholine receptor in a ω-transaminase-catalyzed conversion. A model based on a one-dimensional convolutional neural network was developed to translate raw data augmented Raman spectra directly into substrate concentrations, with which the conversion from ketone to amine by ω-transaminase could be determined over time. The model showed very good predictive capabilities, with R2 values higher than 0.99 for the spectra included in the modeling and 0.964 for an independent dataset. However, the model could not extrapolate outside the concentrations specified by the model. The presented work shows the potential of Raman spectroscopy as a real-time monitoring tool for biocatalytic reactions.

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Candida antarctica lipase B performance in organic solvent at varying water activities studied by molecular dynamics simulations.

Applications of lipases in low-water environments are found across a broad range of industries, including the pharmaceutical and oleochemical sectors. This includes condensation reactions in organic solvents where the enzyme activity has been found to depend strongly on both the solvent and the water activity (aw). Despite several experimental and computational studies, knowledge is largely empirical, and a general predictive approach is much needed. To close this gap, we chose native Candida antarctica lipase B (CALB) and two mutants thereof and used molecular dynamics (MD) simulations to gain a molecular understanding of the effect of aw on the specific activity of CALB in hexane. Based on the simulations, we propose four criteria to understand the performance of CALB in organic media, which is supported by enzyme kinetics experiments. First, the lipase must be stable in the organic solvent, which was the case for native CALB and the two mutants studied here. Secondly, water clusters that form and grow close to the active site must not block the path of substrate molecules into the active site. Thirdly, the lipase's lid must not cover the active site. Finally, mutations and changes in aw must not disrupt the geometry of the active site. We show that mutating specific residues close to the active site can hinder water cluster formation and growth, making the lipase resistant to changes in aw. Our computational screening criteria could potentially be used to screen in-silico designed variants, so only promising candidates could be pushed forward to characterisation.

Toward Kilogram-Scale Peroxygenase-Catalyzed Oxyfunctionalization of Cyclohexane.

Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H2O2 as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h-1, approx. 15 g L-1 h-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation.

Characterization of tyrosine ammonia lyases from Flavobacterium johnsonian and Herpetosiphon aurantiacus.

p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.

EnzymeML: seamless data flow and modeling of enzymatic data.

The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org .

Combining genetic engineering and bioprocess concepts for improved phenylpropanoid production.

The group of natural aromatic compounds known as phenylpropanoids has diverse applications, but current methods of production which are largely based on synthesis from petrochemicals or extraction from agricultural biomass are unsustainable. Bioprocessing is a promising alternative, but improvements in production titers and rates are required to make this method profitable. Here the recent advances in genetic engineering and bioprocess concepts for the production of phenylpropanoids are presented for the purpose of identifying successful strategies, including adaptive laboratory evolution, enzyme engineering, in-situ product removal, and biocatalysis. The pros and cons of bacterial and yeast hosts for phenylpropanoid production are discussed, also in the context of different phenylpropanoid targets and bioprocess concepts. Finally, some broad recommendations are made regarding targets for continued improvement and areas requiring specific attention from researchers to further improve production titers and rates.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization.

Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field. One clear reason is that many evaluate immobilization based on only a few experiments that are not always well-designed. In contrast to many other reviews on the subject, here we highlight the pitfalls of using incorrectly designed immobilization protocols and explain why in many cases sub-optimal results are obtained. We also describe solutions to overcome these challenges and come to the conclusion that recent developments in material science, bioprocess engineering and protein science continue to open new opportunities for the future. In this way, enzyme immobilization, far from being a mature discipline, remains as a subject of high interest and where intense research is still necessary to take full advantage of the possibilities.

Ensuring the Sustainability of Biocatalysis.

Biocatalysis offers many attractive features for the synthetic chemist. In many cases, the high selectivity and ability to tailor specific enzyme features via protein engineering already make it the catalyst of choice. From the perspective of sustainability, several features such as catalysis under mild conditions and use of a renewable and biodegradable catalyst also look attractive. Nevertheless, to be sustainable at a larger scale it will be essential to develop processes operating at far higher concentrations of product, and which make better use of the enzyme via improved stability. In this Concept, it is argued that a particular emphasis on these specific metrics is of particular importance for the future implementation of biocatalysis in industry, at a level that fulfills its true potential.

Combining technology with liquid-formulated lipases for in-spec biodiesel production.

Enzymatic biodiesel production has been at the forefront of biofuels research in recent decades because of the significant environmental advantages it offers, while having the potential to be as effective as conventional chemically catalyzed biodiesel production. However, the higher capital cost, longer reaction time, and sensitivity of enzyme processes have restricted their widespread industrial adoption so far. It is also posited that the lack of research to bring the biodiesel product into final specification has scuppered industrial confidence in the viability of the enzymatic process. Furthermore, the vast majority of literature has focused on the development of immobilized enzyme processes, which seem too costly (and risky) to be used industrially. There has been little focus on liquid lipase formulations such as the Eversa Transform 2.0, which is in fact already used commercially for triglyceride transesterification. It is the objective of this review to highlight new research that focuses on bringing enzymatically produced biodiesel into specification via a liquid lipase polishing process, and the process considerations that come with it.

Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

Ionic liquid-based in situ product removal design exemplified for an acetone-butanol-ethanol fermentation.

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid-liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone-butanol-ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.

High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Towards the sustainable production of bulk-chemicals using biotechnology.

The design and development of new routes for the production of sustainable bulk-chemicals requires focus on feedstock, conversion technology and downstream product recovery. This brief article discusses some of the constraints with using fermentation and suggests the removal of some constraints by using microbial biocatalysis or enzyme biocatalysis, which give a number of benefits in the context of the requirements for bulk-chemical production. Some potential process concepts are described, for products in the suitable low-price range. These examples (biodiesel, furfurals and amines) are used to illustrate the power of biocatalysis. Suggestions for future research efforts beyond molecular biology, involving process-based concepts, are also discussed.

High-level heterologous expression of active Chaetomium thermophilum FDH in Pichia pastoris.

Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications.

Effective removal of antibiotic resistance genes and potential links with archaeal communities during vacuum-type composting and positive-pressure composting.

As a major reservoir of antibiotics, animal manure contributes a lot to the augmented environmental pressure of antibiotic resistance genes (ARGs). This might be the first study to explore the effects of different ventilation types on the control of ARGs and to identify the relationships between archaeal communities and ARGs during the composting of dairy manure. Several ARGs were quantified via Real-time qPCR and microbial communities including bacteria and archaea were analyzed by High-throughput sequencing during vacuum-type composting (VTC) and positive-pressure composting (PPC). The total detected ARGs and class I integrase gene (intI1) under VTC were significantly lower than that under PPC during each stage of the composting (p<0.001). The relative abundance of potential human pathogenic bacteria (HPB) which were identified based on sequencing information and correlation analysis decreased by 74.6% and 91.4% at the end of PPC and VTC, respectively. The composition of archaeal communities indicated that methane-producing archaea including Methanobrevibacter, Methanocorpusculum and Methanosphaera were dominant throughout the composting. Redundancy analysis suggested that Methanobrevibacter and Methanocorpusculum were positively correlated with all of the detected ARGs. Network analysis determined that the possible hosts of ARGs were different under VTC and PPC, and provided new sights about potential links between archaea and ARGs. Our results showed better performance of VTC in reducing ARGs and potential HPB and demonstrated that some archaea could also be influential hosts of ARGs, and caution the risks of archaea carrying ARGs.

Considerations when Measuring Biocatalyst Performance.

As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional measurement techniques focus on a single performance metric (such as the total turnover number), here, it is argued that three metrics (achievable product concentration, productivity, and enzyme stability) are required for an accurate assessment of scalability.

Use of image analysis to understand enzyme stability in an aerated stirred reactor.

Efficient regeneration of NAD(P)+ cofactors is essential for large-scale application of alcohol dehydrogenases due to the high cost and chemical instability of these cofactors. NAD(P)+ can be regenerated effectively using NAD(P)H oxidases (NOXs) that require molecular oxygen as a cosubstrate. In large-scale biocatalytic processes, agitation and aeration are needed for sufficient oxygen transfer into the liquid phase, both of which have been shown to significantly increase the rate of enzyme deactivation. As such, the aim of this study was to identify the existence of a correlation between enzyme stability and gas-liquid interfacial area inside the bioreactor. This was done by measuring gas-liquid interfacial areas inside an aerated stirred reactor, using an in situ optical probe, and simultaneously measuring the kinetic stability of NOXs. Following enzyme incubation at various power inputs and gas-phase compositions, the residual activity was assessed and video samples were analyzed through an image processing algorithm. Enzyme deactivation was found to be proportional to an increase in interfacial area up to a certain limit, where power input appears to have a higher impact. Furthermore, the presence of oxygen increased enzyme deactivation rates at low interfacial areas. The areas were validated with defined glass beads and found to be in the range of those in large-scale bioreactors. Finally, a correlation between the enzyme half-life and specific interfacial area was obtained. Therefore, we conclude that the method developed in this contribution can help to predict the behavior of biocatalyst stability under industrially relevant conditions, concerning specific gas-liquid interfacial areas.

Accelerating the implementation of biocatalysis in industry.

Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed.