Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

The picky mTORC1 in metabolic enzyme degradation.

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.

Statin prevents cancer development in chronic inflammation by blocking interleukin 33 expression.

Chronic inflammation is a major cause of cancer worldwide. Interleukin 33 (IL-33) is a critical initiator of cancer-prone chronic inflammation; however, its induction mechanism by environmental causes of chronic inflammation is unknown. Herein, we demonstrate that Toll-like receptor (TLR)3/4-TBK1-IRF3 pathway activation links environmental insults to IL-33 induction in the skin and pancreas inflammation. An FDA-approved drug library screen identifies pitavastatin to effectively suppress IL-33 expression by blocking TBK1 membrane recruitment/activation through the mevalonate pathway inhibition. Accordingly, pitavastatin prevents chronic pancreatitis and its cancer sequela in an IL-33-dependent manner. The IRF3-IL-33 axis is highly active in chronic pancreatitis and its associated pancreatic cancer in humans. Interestingly, pitavastatin use correlates with a significantly reduced risk of chronic pancreatitis and pancreatic cancer in patients. Our findings demonstrate that blocking the TBK1-IRF3-IL-33 signaling axis suppresses cancer-prone chronic inflammation. Statins present a safe and effective prophylactic strategy to prevent chronic inflammation and its cancer sequela.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

Unravelling the origin of isoprene in the human body-a forty year Odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Remodeling the Homologous Recombination Mechanism of Yarrowia lipolytica for High-Level Biosynthesis of Squalene.

Squalene is a high-value antioxidant with many commercial applications. The use of microbial cell factories to produce squalene as an alternative to plant and animal extracts could meet increasing market demand. Yarrowia lipolytica is an excellent host for squalene production due to its high levels of acetyl-CoA and a hydrophobic environment. However, the need for precise and complicated gene editing has hindered the industrialization of this strain. Herein, the rapid construction of a strain with high squalene production was achieved by enhancing the homologous recombination efficiency in Y. lipolytica. First, remodeling of the homologous recombination efficiency resulted in a 10-fold increase in the homologous recombination rate. Next, the whole mevalonate pathway was integrated into the chromosome to enhance squalene production. Then, a higher level of squalene accumulation was achieved by increasing the level of acetyl coenzyme A and regulating the downstream steroid synthesis pathway. Finally, the squalene production reached 35 g/L after optimizing the fermentation conditions and performing a fed-batch culture in a 5 L jar fermenter. This is the highest squalene production ever reported to date by de novo biosynthesis without adding any inhibitors, paving a new path toward the industrial production of squalene and its downstream products.

Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae.

Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux toward the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7 mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae. ONE-SENTENCE SUMMARY: In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.

Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle).

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/ß conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

pH-dependent reaction triggering in PmHMGR crystals for time-resolved crystallography.

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.

Targeting the mevalonate or Wnt pathways to overcome CAR T-cell resistance in TP53-mutant AML cells.

TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are characterized by chemotherapy resistance and represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cells might be a promising therapeutic option for TP53-mutant AML/MDS. However, the impact of TP53 deficiency in AML cells on the efficacy of CAR T-cells is unknown. We here show that CAR T-cells engaging TP53-deficient leukemia cells exhibit a prolonged interaction time, upregulate exhaustion markers, and are inefficient to control AML cell outgrowth in vitro and in vivo compared to TP53 wild-type cells. Transcriptional profiling revealed that the mevalonate pathway is upregulated in TP53-deficient AML cells under CAR T-cell attack, while CAR T-cells engaging TP53-deficient AML cells downregulate the Wnt pathway. In vitro rational targeting of either of these pathways rescues AML cell sensitivity to CAR T-cell-mediated killing. We thus demonstrate that TP53 deficiency confers resistance to CAR T-cell therapy and identify the mevalonate pathway as a therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising CAR T-cell therapy-enhancing approach for TP53-deficient AML/MDS.

Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

Expanding the structural diversity of terpenes by synthetic biology approaches.

Terpenoids display chemical and structural diversities as well as important biological activities. Despite their extreme variability, the range of these structures is limited by the scope of natural products that canonically derive from interconvertible five-carbon (C5) isoprene units. New approaches have recently been developed to expand their structural diversity. This review systematically explores the combinatorial biosynthesis of noncanonical building blocks via the coexpression of the canonical mevalonate (MVA) pathway and C-methyltransferases (C-MTs), or by using the lepidopteran mevalonate (LMVA) pathway. Unnatural terpenoids can be created from farnesyl diphosphate (FPP) analogs by chemobiological synthesis and terpene cyclopropanation by artificial metalloenzymes (ArMs). Advanced technologies to accelerate terpene biosynthesis are discussed. This review provides a valuable reference for increasing the diversity of valuable terpenoids and their derivatives, as well as for expanding their potential applications.

The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast.

Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. KEY POINTS: • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production.

Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase.

Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and nonsterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.

Beneficial effect of optimizing the expression balance of the mevalonate pathway introduced into the mitochondria on terpenoid production in Saccharomyces cerevisiae.

Terpenoids are used in various industries, and Saccharomyces cerevisiae is a promising microorganism for terpenoid production. Introducing the mevalonate (MVA) pathway into the mitochondria of a strain with an augmented inherent cytosolic MVA pathway increased terpenoid production but also led to the accumulation of toxic pyrophosphate intermediates that negatively affected terpenoid production. We first engineered the inherent MVA pathway in the cytosol and then introduced the MVA pathway into the mitochondria using several promoter combinations, considering the toxicity of pyrophosphate intermediates. However, the highest titer, 183 mg/L, tends to be only 5% higher than that of the strain that only augmented the inherent MVA pathway (SYCM1; 174 mg/L). Next, we hypothesized that, in addition to the toxicity of pyrophosphate, other compounds in the MVA pathway could affect the squalene titer. Thus, we constructed a combinatorial strain library expressing MVA pathway enzymes in the mitochondria with various promoter combinations. The highest squalene titer (230 mg/L) was 32% higher than that of SYCM1. The promoter set revealed that mitigation of mono- and pyrophosphate compound accumulation was important for mitochondrial usage. This study demonstrated that a combinatorial strain library is useful for discovering the optimal gene expression balance in engineering yeast.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Building a machine-learning model to predict optimal mevalonate pathway gene expression levels for efficient production of a carotenoid in yeast.

Simultaneous modification of the expression levels of many metabolic enzyme genes results in diverse expression ratios of these genes; however, the relationship between gene expression levels and chemical productivity remains unclear. However, clarification of this relationship is expected to improve the productivity of useful chemicals. Supervised machine learning is considered to be an effective means to clarify this relationship. In this study, to improve the productivity of carotenoids in yeast Saccharomyces cerevisiae, we aimed to build a machine-learning model that can predict the optimal gene expression level for carotenoid production. First, we obtained data on the expression levels of mevalonate pathway enzyme genes and carotenoid production. Then, based on these data, we built a machine-learning model to predict carotenoid productivity based on gene expression levels. The prediction accuracy of 0.6292 (coefficient of determination) was achieved using the test data. The maximum predicted carotenoid productivity was 4.3 times higher in the engineered strain than in the parental strain, suggesting that the expression levels of the mevalonate pathway enzyme genes tHMG1 and ERG8 have a particularly large impact on carotenoid productivity. This study could be one of the important achievements in addressing the uncertainty of genotype-phenotype correlations, which is one of the challenges facing metabolic engineering strategies.

Pathogenesis-directed treatment of linear porokeratosis with topical cholesterol-lovastatin.

A 2-year-old boy presented with an extensive, asymptomatic, photosensitive eruption refractory to topical steroids and tretinoin; examination and biopsies were consistent with generalized linear porokeratosis involving the face, limbs, and trunk. Treatment with topical cholesterol-lovastatin was initiated, and it successfully improved early erythematous lesions. Whole exome sequencing that targeted mevalonate pathway genes crucial in cholesterol synthesis later revealed a pathogenic, paternally inherited, porokeratosis-associated MVD, c.70+5 G>A, mutation. Topical cholesterol-lovastatin is a safe and effective empiric treatment for porokeratosis when used in the early, erythematous phase, and its success is likely mediated through its role in targeting mevalonate pathway mutations.