Conformational Dynamics in Corynebacterium glutamicum Diaminopimelate Epimerase: Insights from Ligand Parameterization, Atomistic Simulation, and Markov State Modeling.

The microbial enzyme diaminopimelate epimerase (DapF), a vital enzyme in the lysine biosynthetic pathway, catalyzes the conversion of L, L-diaminopimelate (L, L-DAP) to D, L-diaminopimelate (D, L-DAP) using a catalytic cysteine dyad with one cysteine in thiol state and another in thiolate. Under oxidizing conditions, the catalytic cysteines of apo DapF form a disulfide bond that alters the structure and function of DapF. Given its potential as a target for antimicrobial resistance treatments, understanding DapF's functional dynamics is imperative. In the present work, we employ microsecond-scale all-atom molecular dynamics simulations of product-bound DapF and apo-DapF under oxidized and reduced conditions. We employ a polarized charge model for the ligand and the active site residues, which was necessary to preserve the electrostatic environment in the active site and retain the ligand in the active site. The product-bound DapF and apo-DapF in oxidized and reduced conditions exhibit a closed, semi-open, and open conformation, respectively, as identified using the internal coordinates of the dimeric enzyme and the principal component analysis. The conformational switch is guided by the dynamic catalytic (DC) loop, loop II, and loop III movements in the active site. The time scale of the close-to-open conformational transition is estimated to be 0.8 µs through Markov state modeling (MSM) and transition path theory (TPT). The present study explains the role of various active site residues and loops in ligand binding and protein dynamics in the DapF enzyme under different redox conditions. Such information will be helpful in future inhibitor design studies targeting the DapF enzyme.

Microbial chassis design and engineering for production of gamma-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid which is widely applied in agriculture and pharmaceutical additive industries. GABA is synthesized from glutamate through irreversible α-decarboxylation by glutamate decarboxylase. Recently, microbial synthesis has become an inevitable trend to produce GABA due to its sustainable characteristics. Therefore, reasonable microbial platform design and metabolic engineering strategies for improving production of GABA are arousing a considerable attraction. The strategies concentrate on microbial platform optimization, fermentation process optimization, rational metabolic engineering as key metabolic pathway modification, promoter optimization, site-directed mutagenesis, modular transporter engineering, and dynamic switch systems application. In this review, the microbial producers for GABA were summarized, including lactic acid bacteria, Corynebacterium glutamicum, and Escherichia coli, as well as the efficient strategies for optimizing them to improve the production of GABA.

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

Development of a novel, robust and cost-efficient process for valorizing dairy waste exemplified by ethanol production.

BACKGROUND: Delactosed whey permeate (DWP) is a side stream of whey processing, which often is discarded as waste, despite of its high residual content of lactose, typically 10-20%. Microbial fermentation is one of the most promising approaches for valorizing nutrient rich industrial waste streams, including those generated by the dairies. Here we present a novel microbial platform specifically designed to generate useful compounds from dairy waste. As a starting point we use Corynebacterium glutamicum, an important workhorse used for production of amino acids and other important compounds, which we have rewired and complemented with genes needed for lactose utilization. To demonstrate the potential of this novel platform we produce ethanol from lactose in DWP. RESULTS: First, we introduced the lacSZ operon from Streptococcus thermophilus, encoding a lactose transporter and a ß-galactosidase, and achieved slow growth on lactose. The strain could metabolize the glucose moiety of lactose, and galactose accumulated in the medium. After complementing with the Leloir pathway (galMKTE) from Lactococcus lactis, co-metabolization of galactose and glucose was accomplished. To further improve the growth and increase the sugar utilization rate, the strain underwent adaptive evolution in lactose minimal medium for 100 generations. The outcome was strain JS95 that grew fast in lactose mineral medium. Nevertheless, JS95 still grew poorly in DWP. The growth and final biomass accumulation were greatly stimulated after supplementation with NH4+, Mn2+, Fe2+ and trace minerals. In only 24 h of cultivation, a high cell density (OD600 of 56.8 ± 1.3) was attained. To demonstrate the usefulness of the platform, we introduced a plasmid expressing pyruvate decarboxylase and alcohol dehydrogenase, and managed to channel the metabolic flux towards ethanol. Under oxygen-deprived conditions, non-growing suspended cells could convert 100 g/L lactose into 46.1 ± 1.4 g/L ethanol in DWP, a yield of 88% of the theoretical. The resting cells could be re-used at least three times, and the ethanol productivities obtained were 0.96 g/L/h, 2.2 g/L/h, and 1.6 g/L/h, respectively. CONCLUSIONS: An efficient process for producing ethanol from DWP, based on C. glutamicum, was demonstrated. The results obtained clearly show a great potential for this newly developed platform for producing value-added chemicals from dairy waste.

A preliminary study on l-lysine fermentation from lignocellulose feedstock and techno-economic evaluation.

l-Lysine is a commodity amino acid produced from starch feedstock. Various alternative feedstocks had been used for l-lysine production, but the yield was very low. This study took the first preliminary investigation on l-lysine production from lignocellulose for the replacement of food-crop starch. Corn stover was dry acid pretreated and biodetoxified, then used for enzymatic hydrolysis and l-lysine fermentation by an industrial Corynebacterium glutamicum strain. Various fermentation parameters, nutrient additions, and operation variables were applied and finally 33.8 g/L of l-lysine was obtained. This l-lysine titer is still below that of starch based fermentation, but already 3-5 folds greater than that of other alternative feedstocks based fermentation. A techno-economic analysis was conducted and the minimum selling price of l-lysine (hydrochloride form) was calculated to be $2.445 per kg. The cost reduction by the future improvement could fill the technical and economic gap between the cellulosic and starch based l-lysine production.

Rational design of substrate binding pockets in polyphosphate kinase for use in cost-effective ATP-dependent cascade reactions.

Adenosine-5'-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK (Corynebacterium glutamicum) and PPK (Sinorhizobium meliloti) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.

Comprehensive assessment of the L-lysine production process from fermentation of sugarcane molasses.

L-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of L-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to L-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of L-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the L-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g L-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products.

Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae in particular, have become established as important industrial workhorses in biotechnology. Recent years have seen tremendous progress in their advance into tailor-made producers, driven by the upcoming demand for sustainable processes and renewable raw materials. Here, the diversity and complexity of nature is simultaneously a challenge and a benefit. Harnessing biodiversity in the right manner through synergistic progress in systems metabolic engineering and chemical synthesis promises a future innovative bio-economy.

Economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using Corynebacterium glutamicum immobilized in porous polyurethane filler.

An immobilized fermentation system, using cassava bagasse hydrolysate (CBH) and mixed alkalis, was developed to achieve economical succinic acid production by Corynebacterium glutamicum. The C. glutamicum strains were immobilized in porous polyurethane filler (PPF). CBH was used efficiently as a carbon source instead of more expensive glucose. Moreover, as a novel method for regulating pH, the easily decomposing NaHCO3 was replaced by mixed alkalis (NaOH and Mg(OH)2) for succinic acid production by C. glutamicum. Using CBH and mixed alkalis in the immobilized batch fermentation system, succinic acid productivity of 0.42gL(-1)h(-1) was obtained from 35gL(-1) glucose of CBH, which is similar to that obtained with conventional free-cell fermentation with glucose and NaHCO3. In repeated batch fermentation, an average of 22.5gL(-1) succinic acid could be obtained from each batch, which demonstrated the enhanced stability of the immobilized C. glutamicum cells.

Rapid assessment of oxygen transfer impact for Corynebacterium glutamicum.

Oxygen supply is crucial in industrial application of microbial systems, such as Corynebacterium glutamicum, but oxygen transfer is often neglected in early strain characterizations, typically done under aerobic conditions. In this work, a new procedure for oxygen transfer screening is presented, assessing the impact of maximum oxygen transfer conditions (OTRmax) within microtiter plate-based cultivation for enhanced throughput. Oxygen-dependent growth and productivity were characterized for C. glutamicum ATCC13032 and C. glutamicum DM1933 (lysine producer). Biomass and lysine product yield are affected at OTRmax below 14 mmol L(-1) h(-1) in a standardized batch process, but not by further increase of OTRmax above this threshold value indicating a reasonable tradeoff between power input and oxygen transfer capacity OTRmax. The described oxygen transfer screening allows comparative determination of metabolic robustness against oxygen transfer limitation and serves identification of potential problems or opportunities later created during scale-up.

Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. L-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, L-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C. glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.

[Cost-effective production of protein by using cellulose-binding domain fusion tag in Corynebacterium glutamicum].

The CBD gene from Trichoderma reesei was cloned into the Corynebacterium glutamicum secretion expression vector pXMJ19-sp, in which green fluorescent protein was inserted to obtain pXMJ19-sp-GFP-CBD. After induced by 0.5 mmol/L IPTG, GFP-CBD was expressed in Corynebacterium glutamicum at high level of 200 mg/L. The GFP-CBD could be purified to high purity with cellulose column. The results indicated CBD can be successfully used in Corynebacterium glutamicum expression system and thus offer an extremely simple, effective and scalable way for production of recombinant proteins.

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing.

BACKGROUND: Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. RESULTS: Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. CONCLUSIONS: This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

A possibilistic framework for constraint-based metabolic flux analysis.

BACKGROUND: Constraint-based models allow the calculation of the metabolic flux states that can be exhibited by cells, standing out as a powerful analytical tool, but they do not determine which of these are likely to be existing under given circumstances. Typical methods to perform these predictions are (a) flux balance analysis, which is based on the assumption that cell behaviour is optimal, and (b) metabolic flux analysis, which combines the model with experimental measurements. RESULTS: Herein we discuss a possibilistic framework to perform metabolic flux estimations using a constraint-based model and a set of measurements. The methodology is able to handle inconsistencies, by considering sensors errors and model imprecision, to provide rich and reliable flux estimations. The methodology can be cast as linear programming problems, able to handle thousands of variables with efficiency, so it is suitable to deal with large-scale networks. Moreover, the possibilistic estimation does not attempt necessarily to predict the actual fluxes with precision, but rather to exploit the available data--even if those are scarce--to distinguish possible from impossible flux states in a gradual way. CONCLUSION: We introduce a possibilistic framework for the estimation of metabolic fluxes, which is shown to be flexible, reliable, usable in scenarios lacking data and computationally efficient.

Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production.

An optimization strategy based on desirability function approach (DFA) together with response surface methodology (RSM) has been used to optimize production medium in L-glutamine fermentation. Fermentation problems often force to reach a compromise between different experimental variables in order to achieve the most suitable strategy applying in industrial production. The importance of the use of multi-objective optimization methods lies in the ability to cope with this kind of problems. A sequential RSM with different combinations of glucose and (NH(4))(2)SO(4) was performed to attain the optimal medium (OM-1) in glutamine production. Based on the result of RSM and the evaluation of production cost, a more economical optimal medium (OM-2) was obtained with the aid of DFA. In DFA study, glutamate, the main by-product in glutamine fermentation as another response was considered. Compared with OM-1 in validated experiment, similar amounts of glutamine were obtained in OM-2 while the concentration of glutamate and the production cost decreased by 53.6 and 7.1%, respectively.

Markov Chain Monte Carlo Algorithm based metabolic flux distribution analysis on Corynebacterium glutamicum.

MOTIVATION: Metabolic flux analysis via a (13)C tracer experiment has been achieved using a Monte Carlo method with the assumption of system noise as Gaussian noise. However, an unbiased flux analysis requires the estimation of fluxes and metabolites jointly without the restriction on the assumption of Gaussian noise. The flux distributions under such a framework can be freely obtained with various system noise and uncertainty models. RESULTS: In this paper, a stochastic generative model of the metabolic system is developed. Following this, the Markov Chain Monte Carlo (MCMC) approach is applied to flux distribution analysis. The disturbances and uncertainties in the system are simplified as truncated Gaussian multiplicative models. The performance in a real metabolic system is illustrated by the application to the central metabolism of Corynebacterium glutamicum. The flux distributions are illustrated and analyzed in order to understand the underlying flux activities in the system. AVAILABILITY: Algorithms are available upon request.