Generic Chemometric Models for Metabolite Concentration Prediction Based on Raman Spectra.

Chemometric models for on-line process monitoring have become well established in pharmaceutical bioprocesses. The main drawback is the required calibration effort and the inflexibility regarding system or process changes. So, a recalibration is necessary whenever the process or the setup changes even slightly. With a large and diverse Raman dataset, however, it was possible to generate generic partial least squares regression models to reliably predict the concentrations of important metabolic compounds, such as glucose-, lactate-, and glutamine-indifferent CHO cell cultivations. The data for calibration were collected from various cell cultures from different sites in different companies using different Raman spectrophotometers. In testing, the developed "generic" models were capable of predicting the concentrations of said compounds from a dilution series in FMX-8 mod medium, as well as from an independent CHO cell culture. These spectra were taken with a completely different setup and with different Raman spectrometers, demonstrating the model flexibility. The prediction errors for the tests were mostly in an acceptable range (<10% relative error). This demonstrates that, under the right circumstances and by choosing the calibration data carefully, it is possible to create generic and reliable chemometric models that are transferrable from one process to another without recalibration.

Non-invasive and time-resolved measurement of the respiration activity of Chinese hamster ovary cells enables prediction of key culture parameters in shake flasks.

BACKGROUND: Shake flasks are frequently used for mammalian cell suspension cultures. For process development and routine culture monitoring, information on culture behavior is needed early on. MAIN METHODS AND MAJOR RESULTS: Here, cell-specific oxygen uptake rates (qO2 ) of two CHO cell lines were determined from shake flask experiments by simultaneous measurement of oxygen transfer rates (OTR) and viable cell concentrations (VCC). For cell line one, qO2 decreased from 2.38·10-10  to 1.02·10-10  mmol cell-1  h-1 during batch growth. For cell line two, qO2 was constant (1.90·10-10  mmol h-1 ). Determined qO2 values were used to calculate the VCC from OTR data. Cumulated oxygen consumption and glucose consumption were correlated for both cell lines and enabled calculation of glucose concentrations from OTR data. IgG producing cell line one had an oxygen demand of ∼15 mmoloxygen gglucose -1 , cell line two consumed ∼5 mmoloxygen gglucose -1 . The established correlations for determination of VCC and glucose were successfully transferred to subsequent cultivations for both cell lines. Combined measurement of the OTR and the carbon dioxide transfer rate enabled quantitative determination of the lactate concentration (production and consumption) without sampling. CONCLUSIONS AND IMPLICATIONS: Taken together, non-invasive measurement of the respiration activity enabled time-resolved determination of key culture parameters for increased process understanding in shake flasks.

Maximizing the stability of metabolic engineering-derived whole-cell biocatalysts.

A systematic and powerful knowledge-based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case-by-case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole-cell biocatalysts. They are still largely missing for whole-cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole-cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole-cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.

Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing.

Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U g CDW-1 ) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.

Maximization of cell viability rather than biocatalyst activity improves whole-cell ω-oxyfunctionalization performance.

It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1. Corresponding products, that is, terminal alcohols, aldehydes, and acids, constitute versatile bifunctional building blocks, which are of special interest for polymer synthesis. It could clearly be shown that extensive dodecanoic acid methyl ester uptake mediated by high AlkL levels leads to whole-cell biocatalyst toxification. Thus, cell viability constitutes the primary factor limiting biocatalyst stability and, as a result, process durability. Hence, a compromise had to be found between low biocatalyst activity due to restricted substrate uptake and poor biocatalyst stability due to AlkL-mediated toxification. This was achieved by the fine-tuning of heterologous alkL expression, which, furthermore, enabled the identification of the alkBGT expression level as another critical factor determining biocatalyst stability. Controlled synthesis of AlkL and reduced alkBGT expression finally enabled an increase of product titers by a factor of 4.3 up to 229 g Lorg-1 in a two-liquid phase bioprocess setup. Clearly, ω-oxyfunctionalization process performance was determined by cell viability and thus biocatalyst stability rather than the maximally achievable specific biocatalyst activity. Biotechnol. Bioeng. 2017;114: 874-884. © 2016 Wiley Periodicals, Inc.

Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius.

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50°C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3g/l of isobutanol was produced from glucose and 0.6g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48h at 50°C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.