The future of self-selecting and stable fermentations

Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production

Quantification of Microbial Robustness in Yeast

Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions

A comparative summary of expression systems for the recombinant production of galactose oxidase

Abstract Background The microbes Escherichia coli and Pichia pastoris are convenient prokaryotic and eukaryotic hosts, respectively, for the recombinant production of proteins at laboratory scales. A comparative study was performed to evaluate a range of constructs and process parameters for the heterologous intra- and extracellular expression of genes encoding the industrially relevant enzyme galactose 6-oxidase (EC 1.1.3.9) from the fungus Fusarium graminearum. In particular, the wild-type galox gene from F. graminearum, an optimized variant for E. coli and a codon-optimized gene for P. pastoris were expressed without the native pro-sequence, but with a His-tag either at the N- or the C-terminus of the enzyme. Results The intracellular expression of a codon-optimized gene with an N-terminal His10-tag in E. coli, using the pET16b+ vector and BL21DE3 cells, resulted in a volumetric productivity of 180 U·L-1·h-1. The intracellular expression of the wild-type gene from F. graminearum, using the pPIC3.5 vector and the P. pastoris strain GS115, was poor, resulting in a volumetric productivity of 120 U·L-1·h-1. Furthermore, this system did not tolerate an N-terminal His10-tag, thus rendering isolation of the enzyme from the complicated mixture difficult. The highest volumetric productivity (610 U·L-1·h-1) was achieved when the wild-type gene from F. graminearum was expressed extracellularly in the P. pastoris strain SMD1168H using the pPICZα-system. A C-terminal His6-tag did not significantly affect the production of the enzyme, thus enabling simple purification by immobilized metal ion affinity chromatography. Notably, codon-optimisation of the galox gene for expression in P. pastoris did not result in a higher product yield (g protein·L-1 culture). Effective activation of the enzyme to generate the active-site radical copper complex could be equally well achieved by addition of CuSO4 directly in the culture medium or post-harvest. Conclusions The results indicate that intracellular production in E. coli and extracellular production in P. pastoris comprise a complementary pair of systems for the production of GalOx. The prokaryotic host is favored for high-throughput screening, for example in the development of improved enzymes, while the yeast system is ideal for production scale-up for enzyme applications.</p

Adaptation during propagation improves Clostridium autoethanogenum tolerance towards benzene, toluene and xylenes during gas fermentation

Benzene, toluene and xylenes (BTX) are a group of compounds detected in many crude syngas mixtures. However, BTX have been identified to negatively affect microorganisms, including acetogenic species that are capable of fermenting syngas into valuable biocommodities. In order to overcome BTX inhibitory effects, we describe stepwise adaptation in Clostridium autoethanogenum that leads to tolerance to up to 0.5 mM benzene, 0.21 mM toluene and 0.07 mM xylenes. This is equivalent to eightfold of that which is found in a wood gasification plant syngas stream. Fully adapted cultures matched growth, acetate and ethanol product concentrations, and CO consumption compared to the control. The results demonstrate an efficient route towards producing a highly tolerant, industrially relevant acetogenic strain

Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes

The protective role of intracellular glutathione in Saccharomyces cerevisiae during lignocellulosic ethanol production

To enhance the competitiveness of industrial lignocellulose ethanol production, robust enzymes and cell factories are vital. Lignocellulose derived streams contain a cocktail of inhibitors that drain the cell of its redox power and ATP, leading to a decrease in overall ethanol productivity. Many studies have attempted to address this issue, and we have shown that increasing the glutathione (GSH) content in yeasts confers tolerance towards lignocellulose inhibitors, subsequently increasing the ethanol titres. However, GSH levels in yeast are limited by feedback inhibition of GSH biosynthesis. Multidomain and dual functional enzymes exist in several bacterial genera and they catalyse the GSH biosynthesis in a single step without the feedback inhibition. To test if even higher intracellular glutathione levels could be achieved and if this might lead to increased tolerance, we overexpressed the genes from two bacterial genera and assessed the recombinants in simultaneous saccharification and fermentation (SSF) with steam pretreated spruce hydrolysate containing 10% solids. Although overexpressing the heterologous genes led to a sixfold increase in maximum glutathione content (18 \ub5mol\ua0gdrycellmass−1) compared to the control strain, this only led to a threefold increase in final ethanol titres (8.5 g\ua0L− 1). As our work does not conclusively indicate the cause-effect of increased GSH levels towards ethanol titres, we cautiously conclude that there is a limit to cellular fitness that could be accomplished via increased levels of glutathione

Exploring Microbial Robustness for a Sustainable and Efficient Bioproduction

Efficient microbial cell factories that produce valuable compounds are gaining increasing interest as one path towards a more sustainable economy. Therefore, there is an increasing need for robust microorganisms which can optimally perform even in harsh and challenging industrial conditions. The identification of robustness traits is crucial to improve the already-existing strains and develop new, better ones. Here, different approaches to study microbial robustness are presented. First, single-cell analysis in a cell population might give some insights on the development of more robust sub-populations. Physiological parameters (such as intracellular pH, fluxes, redox balance, etc.) and morphologic features were monitored with fluorescent biosensors and tagged proteins to study the single-cell status. Moreover, a barcoding technique will be used to discover and underline patterns in the development of population dynamics during the different industrial processes. Furthermore, an objective method to quantify robustness was developed for selection of useful strains and a large dataset was analysed to find predictive parameters for robustness. All together, these tools will give the possibility to identify robustness traits and understand robustness leading to improved industrial strains and processes

Robustness: linking strain design to viable bioprocesses

Microbial cell factories are becoming increasingly popular for the sustainable production of various chemicals. Metabolic engineering has led to the design of advanced cell factories; however, their long-term yield, titer, and productivity falter when scaled up and subjected to industrial conditions. This limitation arises from a lack of robustness – the ability to maintain a constant phenotype despite the perturbations of such processes. This review describes predictable and stochastic industrial perturbations as well as state-of-the-art technologies to counter process variability. Moreover, we distinguish robustness from tolerance and discuss the potential of single-cell studies for improving system robustness. Finally, we highlight ways of achieving consistent and comparable quantification of robustness that can guide the selection of strains for industrial bioprocesses

Microbial robustness 101: tools and applications

Striving for a fossil-free society, bio-production is gaining increasing interest over time. Bioproduction applies microorganisms (bacteria, yeast, fungi) to produce valuable chemicals from different raw materials (plant biomass, waste materials, etc.) and offers sustainable use of side-streams and/or waste streams. Bioproduction suffers from challenges such as poor microbial performance and reproducibility. One key feature in this field is microbial robustness, i.e., the stability of a phenotype (cellular function) when a system is challenged by different perturbations. Microbial robustness, due to its abstract nature, has been poorly studied also due to the lack of tools available. Moreover, being able to include robustness evaluation in the early stages of bioprocess and strain design would facilitate their scaling up from the laboratory- to the industrial scales.Here two tools to explore microbial robustness with some applications and case studies in Saccharomyces cerevisiae are presented. First, a way to quantify the robustness of cellular functions was developed. The robustness coefficient proposed allows comparison between strains and cellular functions in a given perturbation space. This method, based on the Fano factor, is dimensionless, free from arbitrary control conditions and frequency-independent. Second, fluorescent biosensors sensing the intracellular environment were developed into a versatile and easy-to-use toolbox. Such toolbox was used in population studies to identify different physiological responses in different strains exposed to industrially-relevant media and conditions. In the future, it will be implemented in single-cell analysis in microfluidic devices and for studying the formation of subpopulations in large-scale fermentations. All together, these tools will give the possibility to identify robustness traits and mechanisms, allowing for physiological insights that are a foundation for improving industrial strains and process designs

Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures

In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h-1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h-1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells.IMPORTANCELactococcus lactis is used in large quantities by the food and biotechnology industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics, and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge of respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures