Biology subjects

Siewers, V.

Publications and source records attributed to Siewers, V..

13 recordsLinked to original sources

bioRxiv2023

Engineering Yeast to Improve Heterologous Abscisic Acid Production

Abscisic acid (ABA) is a high-value product with agricultural, medical and nutritional applications. We previously constructed an ABA cell factory by expressing the ABA metabolic pathway from Botrytis cinerea in the biotechnological workhorse Saccharomyces cerevisiae. In this study, we aimed to improve ABA production and explored various rational engineering targets mostly focusing on increasing the activity of two rate-limiting cytochrome P450 monooxygenases of the ABA pathway, BcABA1 and BcABA2. We evaluated the effects of overexpression and knock-down of cell membrane transporters, expression of heterologous cytochrome b5, overexpression of a rate-limiting heme biosynthesis gene and overexpression or knock-out of genes involved in ER membrane homeostasis. One of the genes involved in ER membrane homeostasis, PAH1, was identified as the most promising engineering target. Knock-out of PAH1 improved ABA titers, but also caused a sever growth defect. By replacing the PAH1 promoter with a weak minimal promoter, it was possible to mediate the growth defect while still improving ABA production. In this report we were able to improve the ABA cell factory and furthermore provide valuable insights for future studies aiming to engineer cytochrome P450 monooxygenases. One-sentence summaryIn this study we explored various strategies to improve heterologous abscisic acid production in Saccharomyces cerevisiae and identified fine-tuning of the PAH1 gene as a promising engineering strategy.

bioengineering↗

bioRxiv2023

Sense and Screen-ability: Development of tuneable, biosensor-based screening platforms for abscisic acid

The activities of heterologous enzymes often limit the production titers, rates and yields of cell factories. With the help of biosensors, large random mutagenesis libraries can be screened for improved enzyme variants in a high-throughput manner, even if the enzyme-of-interest is poorly characterised. We previously constructed a Saccharomyces cerevisiae cell factory for the heterologous production of abscisic acid (ABA), a high-value product with a broad range of applications in medicine, agriculture and nutrition. In the current study, we developed high-throughput screening platform strains for two rate-limiting cytochrome P450 monooxygenases, BcABA1 and BcABA2, in the ABA biosynthetic pathway. The screening platforms are designed to minimize the occurrence of false positives during screening experiments. We thoroughly characterised two plant protein-based ABA biosensor candidates. Furthermore, we designed a simple genetic switch, based on the thiamine-repressible promoter pTHI4, to regulate the expression level of enzyme variants. We demonstrated that ABA production can be fine-tuned by varying thiamine concentration in the media. In-depth analysis of the platform strains revealed that screening conditions can be optimized by varying thiamine concentration and cultivation time, making it possible to utilize the full dynamic and operational range of the biosensor. In the future, the constructed strains can be used to screen for improved BcABA1 and BcABA2 variants.

bioengineering↗

bioRxiv2022

Fine-tuning of coumaric acid synthesis to increase naringenin production in yeast

(2S)-Naringenin is a key precursor for biosynthesis of various high-value flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2S)-naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2S)-naringenin and related pathway intermediate p-coumaric acid, which is an important factor for efficient production. Here, we show that fine-turning of p-coumaric acid synthesis enables alleviated cell burden and improved (2S)-naringenin production in yeast. First, we systematically optimized the (2S)-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p-coumaric acid and increasing malonyl-CoA supply, which improved (2S)-naringenin production but significant amounts of p-coumaric acid still accumulated outside the cell. We further established a dual dynamic control system through combing a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p-coumaric acid and downregulate a pathway competing for malonyl-CoA. The optimized strains remarkably decreased extracellular accumulation of p-coumaric acid and simultaneously improved (2S)- naringenin production. Finally, production of 933 mg/L of (2S)-naringenin could be achieved by using minimal medium with negligible accumulation of p-coumaric acid. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.

synthetic biology↗

bioRxiv2021

Supervised generative design of regulatory DNA forgene expression control

In order to control gene expression, regulatory DNA variants are commonly designed using random synthetic approaches with mutagenesis and screening. This however limits the size of the designed DNA to span merely a part of a single regulatory region, whereas the whole gene regulatory structure including the coding and adjacent non-coding regions is involved in controlling gene expression. Here, we prototype a deep neural network strategy that models whole gene regulatory structures and generates de novo functional regulatory DNA with prespecified expression levels. By learning directly from natural genomic data, without the need for large synthetic DNA libraries, our ExpressionGAN can traverse the whole sequence-expression landscape to produce sequence variants with target mRNA levels as well as natural-like properties, including over 30% dissimilarity to any natural sequence. We experimentally demonstrate that this generative strategy is more efficient than a mutational one when using purely natural genomic data, as 57% of the newly-generated highly-expressed sequences surpass the expression levels of natural controls. We foresee this as a lucrative strategy to expand our knowledge of gene expression regulation as well as increase expression control in any desired organism for synthetic biology and metabolic engineering applications.

synthetic biology↗

bioRxiv2021

Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0

Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into GEMs was first enabled by the GECKO method, allowing the study of phenotypes constrained by protein limitations. Here, we upgraded the GECKO toolbox in order to enhance models with enzyme and proteomics constraints for any organism with an available GEM reconstruction. With this, enzyme-constrained models (ecModels) for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus were generated, aiming to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions revealed that upregulation and high saturation of enzymes in amino acid metabolism were found to be common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO was further developed by the implementation of an automated framework for continuous and version-controlled update of ecModels, which was validated by producing additional high-quality ecModels for Escherichia coli and Homo sapiens. These efforts aim to facilitate the utilization of ecModels in basic science, metabolic engineering and synthetic biology purposes.

systems biology↗

bioRxiv2021

The yeast eIF2 kinase Gcn2 facilitates H2O2-mediated feedback inhibition of both protein synthesis and ER oxidative folding during recombinant protein production

Recombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, micro-cultivation and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak -amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find Gcn2-dependent phosphorylation of eIF2 to increase alongside translational attenuation in strains engineered to produce large amounts of -amylase. Gcn2 removal significantly improved -amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H2O2 levels and the unfolded protein response whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H2O2. ImportanceReactive oxygen species (ROS) accumulate during recombinant protein production both in yeast and Chinese hamster ovary cells, two of the most popular organisms used in the multi-million dollar protein production industry. Here we document increased H2O2 in the cytosol of yeast cells producing -amylase. Since H2O2 predominantly targets the protein synthesis machinery and activates the translation initiation factor kinase Gcn2, we removed Gcn2, resulting in increased recombinant -amylase production in two different previously engineered high-producing protein production strains. Removal of this negative feed-back loop thus represents a complementary strategy for improving recombinant protein production efforts currently used in yeast. Gcn2-deficiency also increased the expression of antioxidant genes and the ER-foldase PDI1, suggesting that protein synthesis and ER oxidative folding are linked and feed-back regulated via H2O2. Identification of additional components in this complex regulation may further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

bioengineering↗

bioRxiv2020

Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?

Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to our current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive targets. Engineering Saccharomyces cerevisiae to produce alkanes can be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.

synthetic biology↗

bioRxiv2020

A Single Chromosome Strain of S. cerevisiae Exhibits Diminished Ethanol Metabolism and Tolerance.

This study characterized the growth, metabolism, and transcriptional profile of a S. cerevisiae strain with a single large chromosome that was constructed via successive chromosomal fusions. The single chromosome strain exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a wildtype strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed diminished induction of genes involved in diauxic shift, ethanol metabolism, fatty-acid {beta}-oxidation, and methylglyoxal catabolism during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling predicted that decreased flux through these poorly induced enzymes results in diminished ATP formation and decreased biomass accumulation observed. Together, these observations suggest that switch-like control of carbon source dependent gene expression in S. cerevisiae requires genome arrangement into multiple chromosomes.

systems biology↗

bioRxiv2019

Young Genes are More Responsive to Environmental Stress than Ancient Genes in Budding Yeasts

The Saccharomycotina subphylum (budding yeasts) spans more than 400 million years of evolution and includes species that thrive in many of Earths harsh environments. Characterizing species that grow in harsh conditions could enable the design of more robust yeast strains for biotechnology. However, tolerance to stressful conditions is a multifactorial response, which is difficult to understand since many of the genes involved are as yet uncharacterized. In this work, three divergent yeast species were grown under multiple stressful conditions to identify stress-induced genes. For each condition, duplicated and non-conserved genes were significantly enriched for stress responsiveness compared to single-copy conserved genes. To understand this further, we developed a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis of the sets of genes that changed expression revealed a relationship between stress-induced genes and the youngest gene set, regardless of the species or stress in question. These young genes are rarely essential for growth and evolve rapidly, which may facilitate their functionalization for stress tolerance and may explain their stress-induced expression. These findings show that systems-level analyses that consider gene age can expedite the identification of stress tolerance genes.

systems biology↗

bioRxiv2018

Investigation of putative regulatory acetylation sites in Fas2p of Saccharomyces cerevisiae

Yeast metabolism is highly regulated, in part via coordinated reprogramming of metabolism on a transcriptional level, for example in response to environmental changes. Furthermore, regulation occurs on the protein level via posttranslational modifications directly affecting enzymatic activity - a mode of regulation that has the benefit of being very fast in response to environmental changes. One group of posttranslational modification that has been suggested to have a high impact on regulation of metabolism are acetylations. Around 4000 distinct protein acetylation sites have been found in Saccharomyces cerevisiae, many of which are located in central metabolic enzymes. However, reports on the verification of regulatory roles of specific acetylation sites on these metabolic enzymes have yet to emerge. This study investigates putative regulatory acetylation sites on Fas2p, which in concert with Fas1p is responsible for cytosolic fatty acid (FA) biosynthesis in S. cerevisiae. Fas2p stands out as one of the most highly acetylated proteins in yeast and is located at a branchpoint of acetyl-CoA metabolism. The amino acids (AAs) glutamine (Q) and arginine (R) were introduced to mimic a constitutively acetylated or non-acetylatable state at three separate lysine sites (K) (K83, K173 and K1551) confirmed to be acetylated in two independent studies, either separately or simultaneously. The results suggest that the residue replacement system in the specific case interferes with the enzymatic function of the fatty acid synthase (FAS), as QQQ and RRR triple mutants both reduce the amount of secreted free fatty acids (FFAs) in a faa1{Delta} faa4{Delta} yeast deletion mutant. The K173Q substitution significantly decreased C16 FA species at the expense of C18 FAs, while no such change could be observed for the corresponding K173R modification.

bioengineering↗

bioRxiv2018

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the shape index, a characteristic feature of each TSS describing the spatial distribution of transcription initiation events, has a surprisingly strong negative correlation with the measured expression levels. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

systems biology↗

bioRxiv2018

Engineering Lipid Droplet Assembly Mechanisms for Improved Triacylglycerols Accumulation in S. cerevisiae

Production of triacylglycerols (TAGs) through microbial fermentation is an emerging alternative to plant and animal-derived sources. The yeast Saccharomyces cerevisiae is a preferred organism for industrial use but has natively a very poor capacity of TAG production and storage. Here, we engineered S. cerevisiae for accumulation of high TAG levels through the use of structural and physiological factors that influence assembly and biogenesis of lipid droplets. First, human and fungal perilipin genes were expressed, increasing TAG content by up to 36% when expressing the human perilipin gene PLIN3. Secondly, expression of the FIT2 homologue YFT2 resulted in a 26% increase in TAG content. Lastly, the genes ERD1 and PMR1 were deleted in order to induce an ER stress response and stimulate lipid droplet formation, increasing TAG content by 72% for {Delta}erd1, with an additive effect for both YFT2 and PLIN3 expression. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAG, resulting in improvements of up to 138% over those high-producing strains without any substantial growth effects or abnormal cell morphology. We find that these approaches are not only a major advancement in engineering S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.

bioengineering↗

bioRxiv2017

Quantitative in vivo evaluation of the reverse β-oxidation pathway for fatty acid production in Saccharomyces cerevisiae

Production of fatty acids using engineered Saccharomyces cerevisiae cells is a challenging task in part due to low efficiency of the native fatty acid biosynthesis pathway. One option for improving production efficiency relies on exploring alternative fatty acid production pathways with either improved kinetics, thermodynamics or yield properties.\n\nIn this work, we explored the reverse {beta}-oxidation pathway as an alternative pathway for free fatty acid production. Different gene combinations and analysis methods were tested for assessing pathway efficiency when expressed in the yeast Saccharomyces cerevisiae. Even though different alternatives were tested, quantitative analysis showed no improvement or major change in fatty acid production of the tested strains in our conditions. This lack of improvement suggests that the tested pathway designs and constructs are either nonfunctional in the tested conditions or the resulting strains lack a metabolic driving force that is needed for a functional pathway.\n\nWe conclude that expression of the reverse {beta}-oxidation pathway in S. cerevisiae poses many challenges when compared to expression in bacterial systems. These factors gravely hinder development efforts and success rate for producing fatty acids through this pathway.

bioengineering↗