Chemostat Cultures Research Articles

Simultaneous saccharification and fermentation for d-lactic acid production using a metabolically engineered Escherichia coli adapted to high temperature

BackgroundEscherichia coli JU15 is a metabolically engineered strain capable to metabolize C5 and C6 sugars with a high yield of d-lactic acid production at its optimal growth temperature (37 °C). The simultaneous saccharification and fermentation process allow to use lignocellulosic biomass as a cost-effective and high-yield strategy. However, this process requires microorganisms capable of growth at a temperature close to 50 °C, at which the activity of cellulolytic enzymes works efficiently.ResultsThe thermotolerant strain GT48 was generated by adaptive laboratory evolution in batch and chemostat cultures under temperature increments until 48 °C. The strain GT48 was able to grow and ferment glucose to d-lactate at 47 °C. It was found that a pH of 6.3 conciliated with GT48 growth and cellulase activity of a commercial cocktail. Hence, this pH was used for the SSF of a diluted acid-pretreated corn stover (DAPCS) at a solid load of 15% (w/w), 15 FPU/g-DAPCS, and 47 °C. Under such conditions, the strain GT48 exhibited remarkable performance, producing d-lactate at a level of 1.41, 1.42, and 1.48-fold higher in titer, productivity, and yield, respectively, compared to parental strain at 45 °C.ConclusionsIn general, our results show for the first time that a thermal-adapted strain of E. coli is capable of being used in the simultaneous saccharification and fermentation process without pre-saccharification stage at high temperatures.

Quantitative proteomics reveals oxygen-induced adaptations in Caldalkalibacillus thermarum TA2.A1 microaerobic chemostat cultures.

The thermoalkaliphile Caldalkalibacillus thermarum possesses a highly branched respiratory chain. These primarily facilitate growth at a wide range of dissolved oxygen levels. The aim of this study was to investigate the regulation of C. thermarum respiratory chain. C. thermarum was cultivated in chemostat bioreactors with a range of oxygen levels (0.25% O2-4.2% O2). Proteomic analysis unexpectedly showed that both the type I and the type II NADH dehydrogenase present are constitutive. The two terminal oxidases detected were the cytochrome c:oxygen aa 3 oxidase, whose abundance was highest at 4.2% O2. The cytochrome c:oxygen ba 3 oxidase was more abundant at most other O2 levels, but its abundance started to decline below 0.42% O2. We expected this would result in the emergence of the cytochrome c:oxygen bb 3 complex or the menaquinol:oxygen bd complex, the other two terminal oxidases of C. thermarum; but neither was detected. Furthermore, the sodium-proton antiporter complex Mrp was downregulated under the lower oxygen levels. Normally, in alkaliphiles, this enzyme is considered crucial for sodium homeostasis. We propose that the existence of a sodium:acetate exporter decreases the requirement for Mrp under strong oxygen limitation.

Identification and analysis of the key genes for Escherichia coli heterologous protein expression by transcriptomic profiling.

Escherichia coli is a frequently used host for heterologous protein expression, but its expression efficiency is hindered by several limitations, such as formation of inclusion bodies and proteolytic degradation. In this study, we employed high-density fermentation of heterologous protein production in a 5-L bioreactor, resulting in a yield 2.25 times higher than that of the control group. Transcriptional analysis was conducted at three time points after induction for 0h, 4h, and 12h, revealing 420, 301, and 570 upregulated differentially expressed genes, as well as 424, 202, and 525 downregulated genes, respectively. By conducting enrichment analysis, we constructed strains that relieved without iron limitation, exhibiting a 36% increase in biomass and a 32% increase in protein expression. Furthermore, no overflow metabolism of acetic acid was detected during the protein expression process when utilizing chemostat culture, which indicated that the utilization efficiency of glucose was significantly enhanced without iron limitation. This study presents a novel approach to better comprehend the mechanism of high-yield production of heterologous proteins in Escherichia coli.

Exclusive enteral nutrition initiates individual protective microbiome changes to induce remission in pediatric Crohn’s disease

Exclusive enteral nutrition (EEN) is a first-line therapy for pediatric Crohn's disease (CD), but protective mechanisms remain unknown. We established a prospective pediatric cohort to characterize the function of fecal microbiota and metabolite changes of treatment-naive CD patients in response to EEN (German Clinical Trials DRKS00013306). Integrated multi-omics analysis identified network clusters from individually variable microbiome profiles, with Lachnospiraceae and medium-chain fatty acids as protective features. Bioorthogonal non-canonical amino acid tagging selectively identified bacterial species in response to medium-chain fatty acids. Metagenomic analysis identified high strain-level dynamics in response to EEN. Functional changes in diet-exposed fecal microbiota were further validated using gut chemostat cultures and microbiota transfer into germ-free Il10-deficient mice. Dietary model conditions induced individual patient-specific strain signatures to prevent or cause inflammatory bowel disease (IBD)-like inflammation in gnotobiotic mice. Hence, we provide evidence that EEN therapy operates through explicit functional changes of temporally and individually variable microbiome profiles.

Reproducible chemostat cultures to minimize eukaryotic viruses from fecal transplant material

Recent studies indicate an important role of bacteriophages for successful fecal microbiota transplantation (FMT). However, wider clinical applications of FMT are hampered by to donor variability and concerns of infection risks by bacteria and human viruses. To overcome these challenges, mouse cecal and human fecal material were propagated in a chemostat fermentation setup supporting multiplication of bacteria, and phages, while propagation of eukaryotic viruses will be prevented in the absence of eukaryotic host cells. The results showed decrease of the median relative abundance of viral contigs of classified eukaryotic viruses below 0.01%. The corresponding virome profiles showed dilution rate dependency, a reproducibility between biological replicates, and maintained high diversity regarding both the human and mouse inocula. This proof-of-concept cultivation approach may constitute the first step of developing novel therapeutic tools with high reproducibility and with low risk of infection from the donor material to target gut-related diseases.

Adaptive laboratory evolution in a novel parallel shaken pH-auxostat.

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations.

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acidand degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Development strategy of non-GMO organism for increased hemoproteins in Corynebacterium glutamicum: a growth-acceleration-targeted evolution

Heme, found in hemoproteins, is a valuable source of iron, an essential mineral. The need for an alternative hemoprotein source has emerged due to the inherent risks of large-scale livestock farming and animal proteins. Corynebacterium glutamicum, regarded for Qualified Presumption of Safety or Generally Recognized as Safe, can biosynthesize hemoproteins. C. glutamicum single-cell protein (SCP) can be a valuable alternative hemoprotein for supplying heme iron without adversely affecting blood fat levels. We constructed the chemostat culture system to increase hemoprotein content in C. glutamicum SCP. Through adaptive evolution, hemoprotein levels could be naturally increased to address oxidative stress resulting from enhanced growth rate. In addition, we used several specific plasmids containing growth-accelerating genes and the hemA promoter to expedite the evolutionary process. Following chemostat culture for 15 days, the plasmid in selected descendants was cured. The evolved strains showed improved specific growth rates from 0.59 h−1 to 0.62 h−1, 20% enhanced resistance to oxidative stress, and increased heme concentration from 12.95 µg/g-DCW to 14.22–15.24 µg/g-DCW. Notably, the putative peptidyl-tRNA hydrolase-based evolved strain manifested the most significant increase (30%) of hemoproteins. This is the first report presenting the potential of a growth-acceleration-targeted evolution (GATE) strategy for developing non-GMO industrial strains with increased bio-product productivity.

Distinct Short-Term Response of Intracellular Amino Acids in Saccharomyces cerevisiae and Pichia pastoris to Oxidative and Reductive Stress

Despite being frequently encountered, the effect of oxidative or reductive stress on the intracellular metabolism and the response of the intracellular metabolome of yeasts is severely understudied. Non-conventional yeasts are attracting increasing attention due to their large substrate portfolio of non-canonical pathways as well as their production and secretion of proteins. To understand the effects of both stresses on yeast, the conventional model yeast S. cerevisiae and the non-conventional model yeast P. pastoris were perturbed with 5 mM of hydrogen peroxide for oxidative stress and 20 mM of dithiothreitol for reductive stress in well-defined chemostat cultures at a steady state, and fermentation profiles, intracellular amino acid levels, and intracellular glutathione levels were measured. Although stable profiles of extracellular metabolites were observed, significant changes were measured in intracellular amino acid levels within the first five minutes. Collectively, the amino acids ranged from 0.5 to 400 µmol/gDW, with the most significant increase upon the induction of oxidative stress being seen in cysteine (up to 90%) for S. cerevisiae and in aspartate (up to 80%) for P. pastoris. Upon the induction of reductive stress, asparagine nearly halves in S. cerevisiae, while tryptophan decreases by 60% in P. pastoris. By inspecting the time traces of each amino acid, possible mechanisms of pathway kinetics are speculated. This work furthers our understanding of the response of metabolism to oxidative stress in two model yeasts.

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

A Two-Stage Cascade for Increased High-Value Product Accumulation in Chlamydomonas asymmetrica

Cascade systems are used in the large-scale production of astaxanthin, facilitating a successful value-added process despite high accumulating costs. However, their application to other high-value products (HVPs), like lutein, β-carotene, chlorophylls, and fatty acids, remains unexplored. This study investigates Chlamydomonas asymmetica in chemostatic cultures, focusing on the impact of light and dilution rate. A two-stage cascade system is designed, combining high-light growth with low-light pigment accumulation. The results show potential for productivity improvement. Notably, the spacetime yield (STY) of Chlorophyll a increased by 20.96%, reaching 2.73 g·L−1·d−1 at the lowest dilution rate. Lutein maintains a consistent concentration of 22.34 mg·g−1, while β-carotene achieves a maximum STY of 3.60 mg·L·d−1. A cascade modification with a hollow fiber membrane significantly enhances HVP concentrations—Chlorophyll b, Lutein, Chlorophyll a, β-carotene, EPS, and GLA increase 27.23%, 38.95%, 31.88%, 86.19%, 128.7%, and 57.71%, respectively. STY improvements for these HVPs range from 1.78% to 82.96%. This study offers insights into C. asymmetica’s response and proposes a cascade modification for enhanced HVP production and downstream processing efficiency.

Impact of nutrient excess on physiology and metabolism of Sulfolobus acidocaldarius.

Overflow metabolism is a well-known phenomenon that describes the seemingly wasteful and incomplete substrate oxidation by aerobic cells, such as yeasts, bacteria, and mammalian cells, even when conditions allow for total combustion via respiration. This cellular response, triggered by an excess of C-source, has not yet been investigated in archaea. In this study, we conducted chemostat cultivations to compare the metabolic and physiological states of the thermoacidophilic archaeon Sulfolobus acidocaldarius under three conditions, each with gradually increasing nutrient stress. Our results show that S. acidocaldarius has different capacities for the uptake of the two C-sources, monosodium glutamate and glucose. A saturated tricarboxylic acid cycle at elevated nutrient concentrations affects the cell's ability to deplete its intermediates. This includes deploying additional cataplerotic pathways and the secretion of amino acids, notably valine, glycine, and alanine, while glucose is increasingly metabolized via glycogenesis. We did not observe the secretion of common fermentation products, like organic acids. Transcriptomic analysis indicated an upregulation of genes involved in fatty acid metabolism, suggesting the intracellular conservation of energy. Adapting respiratory enzymes under nutrient stress indicated high metabolic flexibility and robust regulatory mechanisms in this archaeon. This study enhances our fundamental understanding of the metabolism of S. acidocaldarius.

KLa based scale-up cultivation of the extremophilic archaeon Sulfolobus acidocaldarius: from benchtop to pilot scale.

The two major scale-up criteria in continuously stirred bioreactors are 1) constant aerated power input per volume (Pg/Vl), and 2) the volumetric O2 mass transfer coefficient (kla). However, Pg/Vl is only influenced by the stirrer geometry, stirrer speed, aeration and working volume, while the kla is additionally affected by physiochemical properties of the medium (temperature, pH, salt content, etc.), sparging of gas and also by the bioreactor design. The extremophilic archaeon Sulfolobus acidocaldarius, thriving at 75°C and pH 3.0, has the potential for many biotechnological applications. However, previous studies imply that the family Sulfolobaceae might be affected by higher oxygen concentration in the headspace (>26%). Hence, adequate oxygen supply without being toxic has to be ensured throughout the different scales. In this study, the scale-up criteria Pg/Vl and kla were analyzed and compared in a 2L chemostat cultivation of S. acidocaldarius on a defined growth medium at 75°C and a pH value of 3.0, using two different types of spargers at the same aerated power input. The scale-up criterion kLa, ensuring a high specific growth rate as well as viability, was then used for scaleup to 20L and 200L. By maintaining a constant kla comparable dry cell weight, specific growth rate, specific substrate uptake rates and viability were observed between all investigated scales. This procedure harbors the potential for further scale-up to industrial size bioreactors.

Microfluidic chemostatic bioreactor for high-throughput screening and sustainable co-harvesting of biomass and biodiesel in microalgae.

As a renewable and sustainable source for energy, environment, and biomedical applications, microalgae and microalgal biodiesel have attracted great attention. However, their applications are confined due to the cost-efficiency of microalgal mass production. One-step strategy and continuous culturing systems could be solutions. However, current studies for optimization throughout microalgae-based biofuel production pipelines are generally derived from the batch culture process. Better tools are needed to study algal growth kinetics in continuous systems. A microfluidic chemostatic bioreactor was presented here, providing low-bioadhesive cultivations for algae in a cooperative environment of gas, nutrition, and temperature (GNT) involved with high throughput. The chip was used to mimic the continuous culture environment of bioreactors. It allowed simultaneously studying of 8×8 different chemostatic conditions on algal growth and oil production in parallel on a 7×7cm2 footprint. On-chip experiments of batch and continuous cultures of Chlorella. sp. were performed to study growth and lipid accumulation under different nitrogen concentrations. The results demonstrated that microalgal cultures can be regulated to grow and accumulate lipids concurrently, thus enhancing lipid productivity in one step. The developed on-chip culturing condition screening, which was more suitable for continuous bioreactor, was achieved at a half shorter time, 64-times higher throughput, and less reagent consumption. It could be used to establish chemostat cultures in continuous bioreactors which can dramatically accelerate the development of renewable and sustainable algal for CO2 fixation and biosynthesis and related systems for advanced sustainable energy, food, pharmacy, and agriculture with enormous social and ecological benefits.

Simultaneous sulfide and methane oxidation by an extremophile

Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum.

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions-hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950)-directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes

BackgroundAnaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures.ResultsIn slow-growing anaerobic chemostat cultures (D = 0.05 h−1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h−1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h−1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h−1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower.ConclusionsAcetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Abolishing storage lipids induces protein misfolding and stress responses in Yarrowia lipolytica.

Yarrowia lipolytica naturally saves excess carbon as storage lipids. Engineering efforts allow redirecting the high precursor flux required for lipid synthesis toward added-value chemicals such as polyketides, flavonoids, and terpenoids. To redirect precursor flux from storage lipids to other products, four genes involved in triacylglycerol and sterol ester synthesis (DGA1, DGA2, LRO1, and ARE1) can be deleted. To elucidate the effect of the deletions on cell physiology and regulation, we performed chemostat cultivations under carbon and nitrogen limitations, followed by transcriptome analysis. We found that storage lipid-free cells show an enrichment of the unfolded protein response, and several biological processes related to protein refolding and degradation are enriched. Additionally, storage lipid-free cells show an altered lipid class distribution with an abundance of potentially cytotoxic free fatty acids under nitrogen limitation. Our findings not only highlight the importance of lipid metabolism on cell physiology and proteostasis, but can also aid the development of improved chassy strains of Y. lipolytica for commodity chemical production.

Enhanced stress resistance of Bifidobacterium breve NRBB57 by induction of stress proteins at near-zero growth rates.

Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.

Progress in fed-batch culture for recombinant protein production in CHO cells.

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells.