Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

Methanol bioconversion into C3, C4, and C5 platform chemicals by the yeast Ogataea polymorpha.

BACKGROUND: One carbon (C1) molecules such as methanol have the potential to become sustainable feedstocks for biotechnological processes, as they can be derived from CO2 and green hydrogen, without the need for arable land. Therefore, we investigated the suitability of the methylotrophic yeast Ogataea polymorpha as a potential production organism for platform chemicals derived from methanol. We selected acetone, malate, and isoprene as industrially relevant products to demonstrate the production of compounds with 3, 4, or 5 carbon atoms, respectively. RESULTS: We successfully engineered O. polymorpha for the production of all three molecules and demonstrated their production using methanol as carbon source. We showed that the metabolism of O. polymorpha is well suited to produce malate as a product and demonstrated that the introduction of an efficient malate transporter is essential for malate production from methanol. Through optimization of the cultivation conditions in shake flasks, which included pH regulation and constant substrate feeding, we were able to achieve a maximum titer of 13 g/L malate with a production rate of 3.3 g/L/d using methanol as carbon source. We further demonstrated the production of acetone and isoprene as additional heterologous products in O. polymorpha, with maximum titers of 13.6 mg/L and 4.4 mg/L, respectively. CONCLUSION: These findings highlight how O. polymorpha has the potential to be applied as a versatile cell factory and contribute to the limited knowledge on how methylotrophic yeasts can be used for the production of low molecular weight biochemicals from methanol. Thus, this study can serve as a point of reference for future metabolic engineering in O. polymorpha and process optimization efforts to boost the production of platform chemicals from renewable C1 carbon sources.

Constant fed-batch cultivation with glucose and propionate as co-substrate: A strategy to fine-tune polyhydroxyalkanoates monomeric composition in Pseudomonas spp.

Pseudomonas sp. LFM693 is a 2-methylisocitrate lyase (prpB) disrupted mutant. This enzyme catalyzes a step in the 2-methylcitrate cycle, the only known and described pathway for propionate oxidation in this organism. The affected mutants can efficiently produce PHA containing even and odd-chain length hydroxyalkanoates (HAeven/odd) in the presence of propionate and glucose. In this study, a constant fed-batch configuration was utilized to control the composition of PHA and decrease the toxicity of propionate. The incorporation of HAodd into the copolymer was linear, ranging from 7 to approximately 30 %, and correlated directly with the propionate/glucose molar ratio in the feeding solution. This allowed for the molecular composition of the mclPHA to be fine-tuned with minimum process monitoring and control. The average PHA content was 52 % cell dry weight with a molar composition that favored 3-hydroxyalkanoates containing C8, C9, and C10. The conversion factor of propionate to HAodd varied between 0.36 and 0.53 mol·mol-1 (YHAodd/prop.), which are significantly lower than the theoretical maximum efficiency (1.0 mol·mol-1). These results along with the lack of 2-methylisocitrate as a byproduct provides further support for the evidence that the mutant prpB- is still capable of oxidizing propionate.

Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media.

Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X-ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH4 PO4 × 6H2 O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics.

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol.

Introduction: Lactate has gained increasing attention as a platform chemical, particularly for the production of the bioplastic poly-lactic acid (PLA). While current microbial lactate production processes primarily rely on the use of sugars as carbon sources, it is possible to envision a future where lactate can be produced from sustainable, non-food substrates. Methanol could be such a potential substrate, as it can be produced by (electro)chemical hydrogenation from CO2. Methods: In this study, the use of the methylotrophic yeast Ogataea polymorpha as a host organism for lactate production from methanol was explored. To enable lactate production in Ogataea polymorpha, four different lactate dehydrogenases were expressed under the control of the methanol-inducible MOX promoter. The L-lactate dehydrogenase of Lactobacillus helveticus performed well in the yeast, and the lactate production of this engineered strain could additionally be improved by conducting methanol fed-batch experiments in shake flasks. Further, the impact of different nitrogen sources and the resulting pH levels on production was examined more closely. In order to increase methanol assimilation of the lactate-producing strain, an adaptive laboratory evolution experiment was performed. Results and Discussion: The growth rate of the lactate-producing strain on methanol was increased by 55%, while at the same time lactate production was preserved. The highest lactate titer of 3.8 g/L in this study was obtained by cultivating this evolved strain in a methanol fed-batch experiment in shake flasks with urea as nitrogen source. This study provides a proof of principle that Ogataea polymorpha is a suitable host organism for the production of lactate using methanol as carbon source. In addition, it offers guidance for the engineering of methylotrophic organisms that produce platform chemicals from CO2-derived substrates. With reduced land use, this technology will promote the development of a sustainable industrial biotechnology in the future.

Blood Endotoxin Levels as Biomarker of Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Growing evidence supports a role of gut-derived metabolites in nonalcoholic fatty liver disease (NAFLD), but the relation of endotoxin levels with gut permeability and NAFLD stage remains unclear. This systematic review with meta-analysis aims to provide further insights. METHODS: PubMed, Embase, and Cochrane Library were searched for studies published until January 2022 assessing blood endotoxins in patients with NAFLD. Meta-analyses and univariate/multivariate meta-regression, as well as correlation analyses, were performed for endotoxin values and potential relationships to disease stage, age, sex, parameters of systemic inflammation, and metabolic syndrome, as well as liver function and histology. RESULTS: Forty-three studies were included, of which 34 were used for meta-analyses. Blood endotoxin levels were higher in patients with simple steatosis vs liver-healthy controls (standardized mean difference, 0.86; 95% confidence interval, 0.62-1.11) as well as in patients with nonalcoholic steatohepatitis vs patients with nonalcoholic fatty liver/non-nonalcoholic steatohepatitis (standardized mean difference, 0.81; 95% confidence interval, 0.27-1.35; P = .0078). Consistently, higher endotoxin levels were observed in patients with more advanced histopathological gradings of liver steatosis and fibrosis. An increase of blood endotoxin levels was partially attributed to a body mass index rise in patients with NAFLD compared with controls. Nevertheless, significant increases of blood endotoxin levels in NAFLD retained after compensation for differences in body mass index, metabolic condition, or liver enzymes. Increases in blood endotoxin levels were associated with increases in C-reactive protein concentrations, and in most cases, paralleled a rise in markers for intestinal permeability. CONCLUSION: Our results support blood endotoxin levels as relevant diagnostic biomarker for NAFLD, both for disease detection as well as staging during disease progression, and might serve as surrogate marker of enhanced intestinal permeability in NAFLD. Registration number in Prospero: CRD42022311166.

Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain's production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.

Designed to Be Green, Economic, and Efficient: A Ketone-Ester-Alcohol-Alkane Blend for Future Spark-Ignition Engines.

Model-based fuel design can tailor fuels to advanced engine concepts while minimizing environmental impact and production costs. A rationally designed ketone-ester-alcohol-alkane (KEAA) blend for high efficiency spark-ignition engines was assessed in a multi-disciplinary manner, from production cost to ignition characteristics, engine performance, ecotoxicity, microbial storage stability, and carbon footprint. The comparison included RON 95 E10, ethanol, and two previously designed fuels. KEAA showed high indicated efficiencies in a single-cylinder research engine. Ignition delay time measurements confirmed KEAA's high auto-ignition resistance. KEAA exhibits a moderate toxicity and is not prone to microbial infestation. A well-to-wheel analysis showed the potential to lower the carbon footprint by 95 percent compared to RON 95 E10. The findings motivate further investigations on KEAA and demonstrate advancements in model-based fuel design.

Data-driven personalization of a physiologically based pharmacokinetic model for caffeine: A systematic assessment.

Physiologically based pharmacokinetic (PBPK) models have been proposed as a tool for more accurate individual pharmacokinetic (PK) predictions and model-informed precision dosing, but their application in clinical practice is still rare. This study systematically assesses the benefit of using individual patient information to improve PK predictions. A PBPK model of caffeine was stepwise personalized by using individual data on (1) demography, (2) physiology, and (3) cytochrome P450 (CYP) 1A2 phenotype of 48 healthy volunteers participating in a single-dose clinical study. Model performance was benchmarked against a caffeine base model simulated with parameters of an average individual. In the first step, virtual twins were generated based on the study subjects' demography (height, weight, age, sex), which implicated the rescaling of average organ volumes and blood flows. The accuracy of PK simulations improved compared with the base model. The percentage of predictions within 0.8-fold to 1.25-fold of the observed values increased from 45.8% (base model) to 57.8% (Step 1). However, setting physiological parameters (liver blood flow determined by magnetic resonance imaging, glomerular filtration rate, hematocrit) to measured values in the second step did not further improve the simulation result (59.1% in the 1.25-fold range). In the third step, virtual twins matching individual demography, physiology, and CYP1A2 activity considerably improved the simulation results. The percentage of data within the 1.25-fold range was 66.15%. This case study shows that individual PK profiles can be predicted more accurately by considering individual attributes and that personalized PBPK models could be a valuable tool for model-informed precision dosing approaches in the future.

A scalable bubble-free membrane aerator for biosurfactant production.

The bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production.

Poly-γ-glutamic acid production by Bacillus subtilis 168 using glucose as the sole carbon source: A metabolomic analysis.

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol-1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a d-glutamic acid monomer content of 49-62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae.

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2-40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO3 , in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, 31 P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.

Methods for the Analysis of Polyphosphate in the Life Sciences.

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function.

An Optimized Ustilago maydis for Itaconic Acid Production at Maximal Theoretical Yield.

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillus terreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Corrigendum: Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

[This corrects the article DOI: 10.3389/fgene.2019.00747.].

Biotechnological upcycling of plastic waste and other non-conventional feedstocks in a circular economy.

The envisaged circular economy requires absolute carbon efficiency and in the long run abstinence from fossil feedstocks, and integration of industrial production with end-of-life waste management. Non-conventional feedstocks arising from industrial production and societal consumption such as CO2 and plastic waste may soon enable manufacture of multiple products from simple bulk chemicals to pharmaceuticals using biotechnology. The change to these feedstocks could be faster than expected by many, especially if the true cost, including the carbon footprint of products, is considered. The efficiency of biotechnological processes can be improved through metabolic engineering, which can help fulfill the promises of the Paris agreement.

Mitochondrial carriers of Ustilago maydis and Aspergillus terreus involved in itaconate production: same physiological role but different biochemical features.

Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis-aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis-aconitate, but malate is well transported only by Um_Mtt1 and 2-oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features.

Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker's yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol-producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl-CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol-producing enzyme fatty acyl-CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

Polyphosphate Chain Length Determination in the Range of Two to Several Hundred P-Subunits with a New Enzyme Assay and 31P NMR.

Currently, 31P NMR is the only analytical method that quantitatively determines the average chain length of long inorganic polyphosphate (>80 P-subunits). In this study, an enzyme assay is presented that determines the average chain length of polyphosphate in the range of two to several hundred P-subunits. In the enzyme assay, the average polyP chain length is calculated by dividing the total polyphosphate concentration by the concentration of the polyphosphate chains. The total polyphosphate is determined by enzymatic polyphosphate hydrolysis with Saccharomyces cerevisiae exopolyphosphatase 1 and S. cerevisiae inorganic pyrophosphatase 1, followed by colorimetric orthophosphate detection. Because the exopolyphosphatase leaves one pyrophosphate per polyphosphate chain, the polyphosphate chain concentration is assayed by coupling the enzymes exopolyphosphatase (polyP into pyrophosphate), ATP sulfurylase (pyrophosphate into ATP), hexokinase (ATP into glucose 6-phosphate), and glucose 6-phosphate dehydrogenase (glucose 6-phosphate into NADPH), followed by fluorometric NADPH detection. The ability of 31P NMR and the enzyme assay to size polyP was demonstrated with polyP lengths in the range from 2 to ca. 280 P-subunits (no polyP with a longer chain length was available). The small deviation between methods (-4 ± 4%) indicated that the new enzyme assay performed accurately. The limitations of 31P NMR (i.e., low throughput, high sample concentration, expensive instrument) are overcome by the enzyme assay that is presented here, which allows for high sample throughput and requires only a commonly available plate reader and micromole per liter concentrations of polyphosphate.

Saccharomyces cerevisiae containing 28% polyphosphate and production of a polyphosphate-rich yeast extract thereof.

Currently, inorganic polyphosphate is chemically synthesized from phosphate rock and added directly to food products. Yeast extract is a concentrate of soluble fractions of Saccharomyces cerevisiae and is, as a food additive, generally regarded as safe. The aim of this study was to biotechnologically produce a naturally polyphosphate-rich yeast extract. Polyphosphate-rich cells were produced with a wild type (non-genetically modified) S. cerevisiae by orthophosphate-starvation and subsequent orthophosphate-feeding, and contained 28% (w/w) polyphosphate (as KPO3) in cell dry weight, which is the highest content reported so far. Four yeast extract production protocols (autolysis, plasmolysis, enzymatic hydrolysis without and with prior heat inactivation) were tested, whereas the latter was the most promising. From the polyphosphate-rich cells, yeast extract paste and powder were produced containing 20% and 14% (w/w, as KPO3) polyphosphate with an average chain length of 31 and 3 P-subunits, 7% and 14% (w/w, as K1.5H1.5PO4) orthophosphate, 22% and 0% (w/w) water, respectively. For the first time, naturally polyphosphate-rich yeast extracts were produced, which possibly can be used as a clean-label food additive and biological alternative to chemically synthesized polyphosphate in food products.