Precision fermentation with mass spectrometry-based spent media analysis.

Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such as those coupled to gas chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.

High cell density cultivation of E. coli in shake flasks for the production of recombinant proteins.

Batch cultivation of recombinant bacteria in shake flasks typically results in low cell density due to nutrient depletion. Previous studies on high cell density cultivation in shake flasks have relied mainly on controlled release mechanisms. Here, we report a true fed-batch strategy to achieve high cell density of recombinant E. coli in shake flasks in 24 h by feeding a mixture of glycerol and yeast extract with a syringe pump. Feed composition and feed rate were obtained by cybernetic model-based, multi-objective optimization. Model parameters were estimated from time-course measurement of substrate, biomass, and dissolved oxygen levels. The optimized process yielded 20.7 g dry cell weight/L, in agreement with the model prediction. Volumetric protein productivity improved by 10-34-fold compared to batch cultivation with 2.8-fold further improvement when the fed-batch process was replicated in a 3 L bioreactor. The process has significance in the routine laboratory cultivations and in scaleup studies.

Process Development for 6-Aminopenicillanic Acid Production Using Lentikats-Encapsulated Escherichia coli Cells Expressing Penicillin V Acylase.

Penicillin V acylase (PVA, EC 3.5.1.11) hydrolyzes the side chain of phenoxymethylpenicillin (Pen V) and finds application in the manufacture of the pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Here, we report the scale-up of cultivation of Escherichia coli whole cells expressing a highly active PVA from Pectobacterium atrosepticum and their encapsulation in polyvinyl alcohol-poly(ethylene glycol) Lentikats hydrogels. A biocatalytic process for the hydrolysis of 2% (w/v) Pen V was set up in a 2 L reactor using the Lentikats-immobilized whole cells, with a customized setup to enable continuous downstream processing of the reaction products. The biocatalytic reaction afforded complete conversion of Pen V for 10 reaction cycles, with an overall 90% conversion up to 50 cycles. The bioprocess was further scaled up to the pilot-scale at 10 L, enabling complete conversion of Pen V to 6-APA for 10 cycles. The 6-APA and phenoxy acetic acid products were recovered from downstream processing with isolated yields of 85-90 and 87-92%, respectively. Immobilization in Lentikats beads improved the stability of the whole cells on storage, maintaining 90-100% activity and similar conversion efficiency after 3 months at 4 °C. The robust PVA biocatalyst can be employed in a continuous process to provide a sustainable route for bulk 6-APA production from Pen V.

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis.

Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD+ as cofactor over NADP+. The purified recombinant P. pini GDH displayed a specific activity of 247.5 U/mg. The enzyme was stable in the pH range 4-8.5 and exhibited excellent thermostability till 50 °C for 24 h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD+-dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2 h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.

Fine-Tuning Native Promoters of Synechococcus elongatus PCC 7942 To Develop a Synthetic Toolbox for Heterologous Protein Expression.

The cyanobacterium Synechococcus elongatus PCC 7942 is a potential photosynthetic cell-factory. In this study, two native promoters from S. elongatus PCC 7942 driving the expression of abundant cyanobacterial proteins phycocyanin (P cpcB7942) and RuBisCO (P rbc7942) were characterized in relation to their sequence features, expression levels, diurnal behavior, and regulation by light and CO2, major abiotic factors important for cyanobacterial growth. P cpcB7942 was repressed under high light intensity, but cultivation at higher CO2 concentration was able to recover promoter activity. On the other hand, P rbc7942 was repressed by elevated CO2 with a negative regulatory region between 300 and 225 bp. Removal of this region flipped the effect of CO2 with Rbc225 being activated only at high CO2 concentration, besides leading to the loss of circadian rhythm. The results from this study on promoter features and regulation will help expand the repertoire of tools for pathway engineering in cyanobacteria.

The effect of CO2 in enhancing photosynthetic cofactor recycling for alcohol dehydrogenase mediated chiral synthesis in cyanobacteria.

The light harvesting photosystem in cyanobacteria offers a potential pathway for the regeneration of the nicotinamide cofactor NADPH, thereby facilitating the application of cyanobacteria as excellent whole cell biocatalysts in oxidoreductase-mediated biotransformation. The use of cyanobacterial metabolism for cofactor recycling improves the atom economy of the process compared to the commonly employed enzyme-coupled cofactor recycling using enzymes such as glucose dehydrogenase. Here we report the asymmetric conversion of acetophenone to chiral 1-phenylethanol by recombinant Synechococcus elongatus PCC 7942 whole cell biocatalyst that expresses the NADPH dependent L. kefir alcohol dehydrogenase. Besides light, it was observed that carbon dioxide levels play a critical role in improving the bioconversion efficiency possibly due to the enhanced growth rate and improved cofactor availability at elevated CO2 levels. Complete reduction of acetophenone to optically pure (R)-1-phenylethanol at 99% enantiomeric excess was achieved within 6 h with a relatively low cell density of 0.66 g/l by coupling optimum light and CO2 levels and without the need for a co-substrate.

Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.