Construction of CRISPR/CAS9 system for industrial Saccharomyces cerevisiae strain and genetic manipulation effect on 2-phenylethanol pathway

Background The market demand for natural 2-phenylethanol (2-PE) continues to increase. Saccharomyces cerevisiae can synthesize 2-PE through the Ehrlich pathway. There are few studies on the improvement of the diploid industrial strains of S. cerevisiae by gene editing technology. There is no report on the comparison of genetic manipulation effect among S.cerevisiae strains with different 2-PE yield background, and the study on knockout of 2-PE downstream product synthesis gene and its effect on the yield of 2-PE have not been found. Results The CRISPR/CAS9 system with high efficiency for diploid S.cerevisiae CWY132 strain for industrial production of 2-PE was constructed. When the length of the homology arm of donor DNA is increased from 60bp to 500bp, the efficiency of gene editing increased from 0–100%. Using CRISPR/CAS9 technology, the branched acetaldehyde dehydrogenase genes ALD2 and ALD3 and the terminal acetyltransferase gene ATF1 in the Ehrlich pathway of S.cerevisiae strains with different 2-PE yields were knocked out. The results showed that in the high-yielding CWY-132 strain, the 2-PE yield decreased from 3.50 g/L to 1.65 g/L when double ALD2 and ALD3 were knocked out, a decrease of 52.8%. When ATF1 was knocked out, the yield of 2-PE decreased to 0.83 g/L, a decrease of 76.2%; In the low-yielding strain PK-2C, the yield of 2-PE increased from 0.21 g/L to 1.20 g/L when ALD2 was knocked out, an increase of 471%. When ATF1 was knocked out, the yield of 2-PE increased to 0.45g/L, an increase of 114%. The results show that the same genetic manipulation strategy for strains with different 2-PE yeilds backgrounds produces significantly different or even opposite effects. In addition, we found that the insufficient supply of NADH in cells can significantly affect the production of 2-PE, and the tolerance of cells to 2-PE is also a key factor that limits the further increase of 2-PE production in high-yielding strain. Conclusions This study shows that the length of the Donor DNA homology arm is a key factor affecting the efficiency of CRISPR/CAS9 gene editing in industrial diploid S. cerevisiae strains. Our result also shows that it is not feasible to increase the 2-PE production in high-yielding strains by blocking the branch pathway in the Ehrlich pathway. Breakthrough in the 2-PE yield of the high-yielding strains requires improved strains’ tolerance to 2-PE and increase the cellular NADH level.

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

Clostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L–1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L–1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae

2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for “natural” 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.

Integrated Multi-Omics Analyses Reveal the Molecular Basis of Tryptophol Over-Accumulation in Saccharomyces Cerevisiae

BackgroundTryptophol (TOL) is a metabolic derivative of tryptophan (Trp) and shows pleiotropic effects in humans, plants and microbes. The mechanisms of TOL biosynthesis were first explored several decades ago. Nonetheless, a systematic interpretation of TOL over-accumulation is still lacking.ResultsBased on TOL yield, a suitable transformation medium (TM1) was used to culture Saccharomyces cerevisiae strain KMLY1-2. The dynamics of TOL production, cell growth, and gene transcription revealed that TOL production was dependent on cell density and the expression of key genes. Additionally, the effects of Trp and phenylalanine (Phe) on TOL production were tested, and the results showed that Trp can significantly facilitate TOL accumulation, but output plateaued (231.02−266.31 mg/L) at Trp concentrations ≥0.6 g/L. In contrast, Phe reduced the stimulatory effect of Trp, which strongly depended on the Phe concentration. To elucidate the molecular basis and regulatory mechanism of TOL overproduction, an integrated analysis of metabolomics, genomics, and transcriptomics was performed. The results revealed that 1) both the Ehrlich pathway and tryptamine-dependent pathway were involved in S. cerevisiae TOL biosynthesis; 2) Trp increased TOL production by enhancing the Ehrlich pathway, in which the steps of transamination (including aminotransferase genes aro9, aat1, bat2 and his5) and decarboxylation (including decarboxylase genes aro10 and pdc5) played important roles. Of course, this process was assisted by amino acid permease genes agp1 and tat2, dihydrolipoyl dehydrogenase gene lpd1, and transcriptional activator gene aro80, etc.; 3) Phe restricted TOL biosynthesis by repressing the transcript levels of genes such as aat1, his5, aro10, pdc5 and aro80, thus interfering with the transamination and decarboxylation reactions; and 4) under sufficient Trp conditions, the de novo Trp biosynthetic pathway and central carbon metabolism (glycolysis, pentose phosphate pathway, and citrate cycle) of S. cerevisiae were weakened, while the content of some amino acids increased, which may be related to the promotion of yeast cell growth by Trp.ConclusionsIn this study, TOL production of S. cerevisiae was significantly improved, and our integrated multi-omics analyses have provided insights into the understanding of TOL over-accumulation, which will be useful for future production of TOL using metabolic engineering strategies.

Curation and Analysis of a Saccharomyces cerevisiae Genome-Scale Metabolic Model for Predicting Production of Sensory Impact Molecules under Enological Conditions

One approach for elucidating strain-to-strain metabolic differences is the use of genome-scale metabolic models (GSMMs). To date GSMMs have not focused on the industrially important area of flavor production and, as such; do not cover all the pathways relevant to flavor formation in yeast. Moreover, current models for Saccharomyces cerevisiae generally focus on carbon-limited and/or aerobic systems, which is not pertinent to enological conditions. Here, we curate a GSMM (iWS902) to expand on the existing Ehrlich pathway and ester formation pathways central to aroma formation in industrial winemaking, in addition to the existing sulfur metabolism and medium-chain fatty acid (MCFA) pathways that also contribute to production of sensory impact molecules. After validating the model using experimental data, we predict key differences in metabolism for a strain (EC 1118) in two distinct growth conditions, including differences for aroma impact molecules such as acetic acid, tryptophol, and hydrogen sulfide. Additionally, we propose novel targets for metabolic engineering for aroma profile modifications employing flux variability analysis with the expanded GSMM. The model provides mechanistic insights into the key metabolic pathways underlying aroma formation during alcoholic fermentation and provides a potential framework to contribute to new strategies to optimize the aroma of wines.

Isotopic Tracers Unveil Distinct Fates for Nitrogen Sources during Wine Fermentation with Two Non-Saccharomyces Strains

Non-Saccharomyces yeast strains have become increasingly prevalent in the food industry, particularly in winemaking, because of their properties of interest both in biological control and in complexifying flavour profiles in end-products. However, unleashing the full potential of these species would require solid knowledge of their physiology and metabolism, which is, however, very limited to date. In this study, a quantitative analysis using 15N-labelled NH4Cl, arginine, and glutamine, and 13C-labelled leucine and valine revealed the specificities of the nitrogen metabolism pattern of two non-Saccharomyces species, Torulaspora delbrueckii and Metschnikowia pulcherrima. In T. delbrueckii, consumed nitrogen sources were mainly directed towards the de novo synthesis of proteinogenic amino acids, at the expense of volatile compounds production. This redistribution pattern was in line with the high biomass-producer phenotype of this species. Conversely, in M. pulcherrima, which displayed weaker growth capacities, a larger proportion of consumed amino acids was catabolised for the production of higher alcohols through the Ehrlich pathway. Overall, this comprehensive overview of nitrogen redistribution in T. delbrueckii and M. pulcherrima provides valuable information for a better management of co- or sequential fermentation combining these species with Saccharomyces cerevisiae.

Enhanced 2-phenylethanol production by newly isolated Meyerozyma sp. strain YLG18 and characterization of its synthetic pathways

Background2-Phenylethanol (2-PE) is an aromatic alcohol which has been widely used in cosmetics, perfume and food industries owning to its delicate rose scent. The newly isolated yeast Meyerozyma guilliermondii YLG18 was able to tolerate high exogenous 2-PE and produce 2-PE with two different pathways.ResultsA unique Meyerozyma sp. strain YLG18 was obtained in this study, which was capable of tolerating 4.0 g/L exogenous 2-PE. Response surface methodology (RSM) was implemented to improve the maximum 2-PE production. At optimized conditions: temperature, 24.7℃; initial glucose, 63.54 g/L; initial L-phe, 10.70 g/L, 2-PE production was increased to 2.55 g/L, leading to 104% increase compared to the pre-optimized one. In situ product recovery (ISPR) could further help improve the final 2-PE production to 3.20 g/L with fatty acid methyl ester as the extractant, representing the highest 2-PE production by using Meyerozyma sp.. Furthermore, genes involved in 2-PE synthesis were identified and their expression levels between Shikimate pathway and Ehrlich pathway were compared. Based on the genomic and transcriptional analysis, a penta-functional enzyme AroM in Shikimate pathway and an aspartate aminotransferase (AAT) with the potential to convert phenylalanine into phenylpyruvate in Ehrlich pathway were identified.ConclusionsThese findings would help broaden our knowledge and add to the pool of known 2-PE generating microbes and genes. Moreover, this study describes a potential, new 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its derivatives in the future. Key words: 2-phenylethanol; Meyerozyma guilliermondii; RSM; ISPR; gene analysis

A yeast platform for high-level synthesis of natural and unnatural tetrahydroisoquinoline alkaloids

ABSTRACTThe tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogues. The plant kingdom manufactures more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a THIQ platform by increasing yeast production of the central BIA intermediate (S)-reticuline to more than 3 g L-1, a 38,000-fold improvement over our first-generation strain. Gains in BIA output coincided with the formation of several substituted THIQs derived from host amino acid catabolism. Enabled by this activity, we repurposed the yeast Ehrlich pathway and demonstrate the synthesis of an array of unnatural THIQ scaffolds. This work provides a blueprint for synthesizing new privileged structures and will enable the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.