Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor.

BACKGROUND: The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. RESULTS: Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L-1 demonstrating the consistency of this study's microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. CONCLUSIONS: The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

Influence of Varying Pre-Culture Conditions on the Level of Population Heterogeneity in Batch Cultures with an Escherichia coli Triple Reporter Strain.

When targeting robust, high-yielding bioprocesses, phenomena such as population heterogeneity have to be considered. Therefore, the influence of the conditions which the cells experience prior to the main culture should also be evaluated. Here, the influence of a pre-culture medium (complex vs. minimal medium), optical density for inoculation of the main culture (0.005, 0.02 and 0.0125) and harvest time points of the pre-culture in exponential growth phase (early, mid and late) on the level of population heterogeneity in batch cultures of the Escherichia coli triple reporter strain G7BL21(DE3) in stirred-tank bioreactors was studied. This strain allows monitoring the growth (rrnB-EmGFP), general stress response (rpoS-mStrawberry) and oxygen limitation (nar-TagRFP657) of single cells through the expression of fluorescent proteins. Data from batch cultivations with varying pre-culture conditions were analysed with principal component analysis. According to fluorescence data, the pre-culture medium had the largest impact on population heterogeneities during the bioprocess. While a minimal medium as a pre-culture medium elevated the differences in cellular growth behaviour in the subsequent batch process, a complex medium increased the general stress response and led to a higher population heterogeneity. The latter was promoted by an early harvest of the cells with low inoculation density. Seemingly, nar-operon expression acted independently of the pre-culture conditions.

Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production.

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.

Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions.

The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.

Advances in automated real-time flow cytometry for monitoring of bioreactor processes.

Flow cytometry and its technological possibilities have greatly advanced in the past decade as analysis tool for single cell properties and population distributions of different cell types in bioreactors. Along the way, some solutions for automated real-time flow cytometry (ART-FCM) were developed for monitoring of bioreactor processes without operator interference over extended periods with variable sampling frequency. However, there is still great potential for ART-FCM to evolve and possibly become a standard application in bioprocess monitoring and process control. This review first addresses different components of an ART-FCM, including the sampling device, the sample-processing unit, the unit for sample delivery to the flow cytometer and the settings for measurement of pre-processed samples. Also, available algorithms are presented for automated data analysis of multi-parameter fluorescence datasets derived from ART-FCM experiments. Furthermore, challenges are discussed for integration of fluorescence-activated cell sorting into an ART-FCM setup for isolation and separation of interesting subpopulations that can be further characterized by for instance omics-methods. As the application of ART-FCM is especially of interest for bioreactor process monitoring, including investigation of population heterogeneity and automated process control, a summary of already existing setups for these purposes is given. Additionally, the general future potential of ART-FCM is addressed.

Applications of Coarse-Grained Models in Metabolic Engineering.

Mathematical modeling is a promising tool for better understanding of cellular processes. In recent years, the development of coarse-grained models has gained attraction since these simple models are able to capture and describe a broad range of growth conditions. Coarse-grained models often comprise only two cellular components, a low molecular component as representative for central metabolism and energy generation and a macromolecular component, representing the entire proteome. A framework is presented that presents a strict mass conservative model for bacterial growth during a biotechnological production process. After providing interesting properties for the steady-state solution, applications are presented 1) for a production process of an amino acid and 2) production of a metabolite from central metabolism.

Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations.

Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli.

OBJECTIVE: Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera, yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDP-glucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase. RESULTS: First, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDP-glucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale. CONCLUSION: This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.