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.

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.

Expression of glycerol-3-phosphate acyltransferase increases non-polar lipid accumulation in Nannochloropsis oceanica.

Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae.

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.