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Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Summary Black tea is the most widely consumed tea drink in the world and has consistently been reported to possess anti-aging benefits. However, whether theaflavins, one type of the characteristic phytochemicals in black tea extracts, are involved in regulating aging and lifespan in consumers remains largely unknown. In this study, we show that theaflavins play a beneficial role in preventing age-onset intestinal leakage and dysbiosis, thus delaying aging in Drosophila . Mechanistically, theaflavins regulate the condensate assembly of Imd to negatively govern the overactivation of Imd signals in fruit fly intestines. In addition, theaflavins prevent DSS-induced colitis in mice, suggesting theaflavins play a role in modulating intestinal integrity. Overall, our study reveals a molecular mechanism by which theaflavins regulate gut homeostasis likely through controlling Imd coalescence.

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of novel and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes, thus determining the fitness effect of disrupted genes. This is accomplished by calculating an optimization metric that quantifies the tradeoff between guide activity and library coverage, which is maximized to accurately classify genes essential to screening conditions. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in gain- and loss-of-function screens under high salt and low pH conditions to identify known and novel genes that were related to stress tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae , this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 ​°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P SSA3 and P ADH1 , increased expression above 40 ​°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 ​°C. Cultures at 41 ​°C exhibited a similar TAL biosynthesis capability, while at 30 ​°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.