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.

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.

Mix and Match: Promoters and Terminators for Tuning Gene Expression in the Methylotrophic Yeast Ogataea polymorpha.

The yeast Ogataea polymorpha is an upcoming host for bio-manufacturing due to its unique physiological properties, including its broad substrate spectrum, and particularly its ability to utilize methanol as the sole carbon and energy source. However, metabolic engineering tools for O. polymorpha are still rare. In this study we characterized the influence of 6 promoters and 15 terminators on gene expression throughout batch cultivations with glucose, glycerol, and methanol as carbon sources as well as mixes of these carbon sources. For this characterization, a short half-life Green Fluorescent Protein (GFP) variant was chosen, which allows a precise temporal resolution of gene expression. Our promoter studies revealed how different promoters do not only influence the expression strength but also the timepoint of maximal expression. For example, the expression strength of the catalase promoter (pCAT) and the methanol oxidase promoter (pMOX) are comparable on methanol, but the maximum expression level of the pCAT is reached more than 24 h earlier. By varying the terminators, a 6-fold difference in gene expression was achieved with the MOX terminator boosting gene expression on all carbon sources by around 50% compared to the second-strongest terminator. It was shown that this exceptional increase in gene expression is achieved by the MOX terminator stabilizing the mRNA, which results in an increased transcript level in the cells. We further found that different pairing of promoters and terminators or the expression of a different gene (ß-galactosidase gene) did not influence the performance of the genetic parts. Consequently, it is possible to mix and match promoters and terminators as independent elements to tune gene expression in O. polymorpha.

An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

Genome-scale model reconstruction of the methylotrophic yeast Ogataea polymorpha.

BACKGROUND: Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. RESULTS: To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. CONCLUSION: The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.

Controlling the Production of Pseudomonas Phenazines by Modulating the Genetic Repertoire.

Microbial phenazines are getting increasing attention for antimicrobial and biotechnological applications. Phenazine production of the most well-known producer Pseudomonas aeruginosa is subject to a highly complex regulation network involving both quorum sensing and catabolite repression. These networks affect the expression of the two redundant phz gene operons responsible for phenazine-1-carboxylate (PCA) production and two specific genes phzM and phzS necessary for pyocyanin production. To decipher the specific functionality of these genes, in this study, specific phenazine gene deletion mutants of P. aeruginosa PA14 were generated and characterized in glucose and 2,3-butanediol media. Phenazine concentration and expression levels of the remaining genes were analyzed in parallel experiments. The findings suggest a strong dominance of operon phzA2-G2 resulting in a 10-fold higher expression of phz2 compared to phzA1-G1 and almost exclusive production of PCA from this operon. The genes phzM and phzS seem to exhibit antagonistic function in phenazine production. An upregulation of phzM explains the documented enhanced pyocyanin production in a 2,3-butanediol medium. Applied to a bioelectrochemical system, the altered phenazine production of the mutant strains is directly translated into current generation. Additionally, the deletion of the phenazine genes induced the activation of alternative energy pathways, which resulted in the accumulation of various fermentation products. Overall, modulating the genetic repertoire of the phenazine genes tremendously affects phenazine production levels, which are naturally kept in tight homeostasis in the P. aeruginosa wildtype. This important information can be directly utilized for ongoing efforts of heterologous biotechnological phenazine production.

Microbial Electrosynthesis I: Pure and Defined Mixed Culture Engineering.

In the past 6 years, microbial bioelectrochemistry has strongly increased in attraction and audience when expanding from mainly environmental technology applications to biotechnology. In particular, the promise to combine electrosynthesis with microbial catalysis opens attractive approaches for new sustainable redox-cofactor recycling, redox-balancing, or even biosynthesis processes. Much of this promise is still not fulfilled, but it has opened and fueled entirely new research areas in this discipline. Activities in designing, tailoring, and applying specific microbial catalysts as pure or defined co-cultures for defined target bioproductions are greatly accelerating. This chapter gives an overview of the current progress as well as the emerging trends in molecular and ecological engineering of defined microbial biocatalysts to prepare them for evolving microbial electrosynthesis processes. In addition, the multitude of microbial electrosynthetic processes with complex undefined mixed cultures is covered by ter Heijne et al. (Adv Biochem Eng Biotechnol. https://doi.org/10.1007/10_2017_15 , 2017). Graphical Abstract.

Boosting mediated electron transfer in bioelectrochemical systems with tailored defined microbial cocultures.

Bioelectrochemical systems (BES) hold great promise for sustainable energy generation via a microbial catalyst from organic matter, for example, from wastewater. To improve current generation in BES, understanding the underlying microbiology of the electrode community is essential. Electron mediator producing microorganism like Pseudomonas aeruginosa play an essential role in efficient electricity generation in BES. These microbes enable even nonelectroactive microorganism like Enterobacter aerogenes to contribute to current production. Together they form a synergistic coculture, where both contribute to community welfare. To use microbial co-operation in BES, the physical and chemical environments provided in the natural habitats of the coculture play a crucial role. Here, we show that synergistic effects in defined cocultures of P. aeruginosa and E. aerogenes can be strongly enhanced toward high current production by adapting process parameters, like pH, temperature, oxygen demand, and substrate requirements. Especially, oxygen was identified as a major factor influencing coculture behavior and optimization of its supply could enhance electric current production over 400%. Furthermore, operating the coculture in fed-batch mode enabled us to obtain very high current densities and to harvest electrical energy for 1 month. In this optimized condition, the coulombic efficiency of the process was boosted to 20%, which is outstanding for mediator-based electron transfer. This study lays the foundation for a rationally designed utilization of cocultures in BES for bioenergy generation from specific wastewaters or for bioprocess sensing and for benefiting from their synergistic effects under controlled bioprocess condition.

Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii.

Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction.

Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440.

Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems (BES). We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 µg/mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 µA/cm(2) anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains.

The acute phase protein haptoglobin and its relation to oxidative status in piglets undergoing weaning-induced stress.

Haptoglobin (Hp) prevents the hemoglobin driven generation of hydroxyl radicals and lipid peroxides. Hp can reduce the neutrophil respiratory burst and is an antioxidative molecule in its own right. We aimed to evaluate Hp concentrations, oxidative stress and antioxidative capacity in blood during weaning and to characterise potential relationships between these parameters. Two batches of 10 piglets each (2 trials) weaned at the age of 27-30 days were fed a starter feed mix ad libitum. Blood samples were taken 1 week before weaning and at weekly intervals thereafter. Oxidative stress was monitored via the D-ROM system, antioxidative capacity was measured with the TEAC assay and Hp concentrations were measured by ELISA. Neutrophil phagocytic activity and oxidative burst were examined via flow-cytometry. Body weights were recorded weekly. Hp concentrations were increased in both trials post-weaning (P < 0.01); oxidative stress and oxidative burst were elevated in trial I (P < 0.005). In trial I, Hp and ROM values returned to baseline levels at 6 weeks post-weaning. The piglets in trial II showed respiratory symptoms and maintained elevated Hp concentrations. ROM values and Hp were related (r = 0.58; P < 0.01). Hp and body weight gain were inversely related post-weaning.