Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid.

BACKGROUND: The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. RESULTS: Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. CONCLUSION: This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.

Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols.

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1S659A/S1157A(ACC1**), as well as the last two steps of TAG formation: phosphatidic phosphatase (PAH1) and diacylglycerol acyltransferase (DGA1), ultimately leading to 129 mg∙gCDW-1 of TAGs. Disruption of TAG lipase genes TGL3, TGL4, TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW-1. Further disruption of the beta-oxidation by deletion of POX1, as well as glycerol-3-phosphate utilization through deletion of GUT2, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW-1. The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.

Engineering lipid droplet assembly mechanisms for improved triacylglycerol accumulation in Saccharomyces 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 endoplasmic reticulum stress response and stimulate lipid droplet formation, increasing TAG content by 72% for Δerd1. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAGs, 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 not only represent a significant improvement of S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion.

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in ß-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae.

BACKGROUND: In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields. RESULTS: We analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain. CONCLUSIONS: Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.

Intragenic recombination has a critical role on the evolution of Legionella pneumophila virulence-related effector sidJ.

SidJ is a Dot/Icm effector involved in the trafficking or retention of ER-derived vesicles to Legionella pneumophila vacuoles whose mutation causes an observable growth defect, both in macrophage and amoeba hosts. Given the crucial role of this effector in L. pneumophila virulence we investigated the mechanisms shaping its molecular evolution. The alignment of SidJ sequences revealed several alleles with amino acid variations that may influence the protein properties. The identification of HGT events and the detection of balancing selection operating on sidJ evolution emerge as a clear result. Evidence suggests that intragenic recombination is an important strategy in the evolutionary adaptive process playing an active role on sidJ genetic plasticity. This pattern of evolution is in accordance with the life style of L. pneumophila as a broad host-range pathogen by preventing host-specialization and contributing to the resilience of the species.