Biological Demands and Toxicity of Isoprenoid Precursors in Bacillus Subtilis Through Cell-Permeant Analogs of Isopentenyl Pyrophosphate and Dimethylallyl Pyrophosphate.

Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

Comparative study of two indoor microbial volatile pollutants, 2-Methyl-1-butanol and 3-Methyl-1-butanol, on growth and antioxidant system of rice (Oryza sativa) seedlings.

2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals.

Stereochemical Divergence of Polyprenol Phosphate Glycosyltransferases.

In the three domains of life, lipid-linked glycans contribute to various cellular processes ranging from protein glycosylation to glycosylphosphatidylinositol anchor biosynthesis to peptidoglycan assembly. In generating many of these glycoconjugates, phosphorylated polyprenol-based lipids are charged with single sugars by polyprenol phosphate glycosyltransferases. The resultant substrates serve as glycosyltransferase donors, complementing the more common nucleoside diphosphate sugars. It had been accepted that these polyprenol phosphate glycosyltransferases acted similarly, given their considerable sequence homology. Recent findings, however, suggest that matters may not be so simple. In this Opinion we propose that the stereochemistry of sugar addition by polyprenol phosphate glycosyltransferases is not conserved across evolution, even though the GT-A fold that characterizes such enzymes is omnipresent.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Metabolomics reveals the role of isopentenyl group in coumarins metabolism.

Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase.

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.

Electrophysiological and Behavioral Responses of an Ambrosia Beetle to Volatiles of its Nutritional Fungal Symbiont.

Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae. During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae, but not antagonistic fungi Beauveria bassiana, Metarhizium brunneum, Trichoderma harzianum, the plant pathogen Fusarium proliferatum, or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae; the latter two compounds were also detected in emissions from B. bassiana. Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016-2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.

Recent advances in the microbial production of isopentanol (3-Methyl-1-butanol).

As the effects of climate change become increasingly severe, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. The design and optimization of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce dependence on fossil fuels and thereby produce fewer emissions. Over the past two decades, a tremendous amount of research has contributed to the development of microbial strains to produce advanced fuel compounds, including branched-chain higher alcohols (BCHAs) such as isopentanol (3-methyl-1-butanol; 3M1B) and isobutanol (2-methyl-1-propanol). In this review, we provide an overview of recent advances in the development of microbial strains for the production of isopentanol in both conventional and non-conventional hosts. We also highlight metabolic engineering strategies that may be employed to enhance product titers, reduce end-product toxicity, and broaden the substrate range to non-sugar carbon sources. Finally, we offer glimpses into some promising future directions in the development of isopentanol producing microbial strains.

Enhancing isoprenoid synthesis in Yarrowia lipolytica by expressing the isopentenol utilization pathway and modulating intracellular hydrophobicity.

The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.

New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions.

BACKGROUND: The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. RESULTS: In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. CONCLUSION: Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

Enriching the Production of 2-Methyl-1-Butanol in Fermentation Process Using Corynebacterium crenatum.

Non-natural 2-methyl-1-butanol (2 MB) has been biosynthesized through the modification of metabolic pathways using Corynebacterium crenatum, a non-model host. However, its production capacity is not effectively improved. In this study, the fermentation process was strengthened through factor combination design (FCD) for enhancing the production of 2 MB. Our results showed that the highest production of 2 MB, 3-methyl-1-butanol (3 MB), ethanol, and total solvent was 4.87 ± 0.39 g/L, 3.57 ± 0.21 g/L, 5.74 ± 0.43 g/L, and 14.18 g/L, respectively, under the optimal fermentation conditions. The optimal fermentation conditions were determined through the FCD to be as follows: pH of 6.5, IPTG concentration of 1.2 mM, fermentation temperature of 32 °C, and fermentation time of 96 h. This study provides a significant guidance for the optimal control technology of the genetically engineered C. crenatum, and also a useful reference for the industrial production of 2 MB via the microbial fermentation approach.

Proteolytic Volatile Profile and Electrophoretic Analysis of Casein Composition in Milk and Cheese Derived from Mironutrient-Fed Cows.

The aim of the study was to evaluate the proteolytic process in Caciocavallo cheese obtained from Friesian cows fed zinc, selenium, and iodine supplementation. Thirty-six Friesian cows, balanced for parity, milk production, and days in milk, were randomly assigned to four groups. The control group (CG) was fed with a conventional feeding strategy, while the three remaining groups received a diet enriched with three different trace elements, respectively zinc (ZG), selenium (SG), and iodine (IG). At the end of the experimental period, samples of milk were collected and used to produce Caciocavallo cheese from each experimental group. Cheese samples were then analyzed after 7 and 120 days from the cheese making in order to obtain information on chemical composition and extent of the proteolytic process, evaluated through the electrophoretic analysis of caseins and the determination of volatiles profile. Both milk and cheese samples were richer in the amount of the microelement respectively used for the integration of the cattle's diet. The zymographic approach was helpful in evaluating, in milk, the proteolytic function performed by endogenous metalloenzymes specifically able to degrade gelatin and casein; this evaluation did not highlight significant differences among the analyzed samples. In cheese, the electrophoretic analysis in reducing and denaturing condition showed the marked ability of ß-casein to resist the proteolytic action during ripening, whereas the dietary selenium supplementation was shown to perform a protective action against the degradation of S1 and S2 isoforms of α-casein. The analysis of the volatile profile evidenced the presence of compounds associated with proteolysis of phenylalanine and leucine. This approach showed that selenium was able to negatively influence the biochemical processes that lead to the formation of 3-methyl butanol, although the identification of the specific mechanism needs further investigation.

Effects of Pheromone Dose and Conspecific Density on the Use of Aggregation-Sex Pheromones by the Longhorn Beetle Phymatodes grandis and Sympatric Species (Coleoptera: Cerambycidae).

Many species of longhorn beetles (Coleoptera: Cerambycidae) utilize male-produced aggregation-sex pheromones that attract both sexes. However, the reasons why and the details of how this type of pheromone is used by cerambycids and other coleopteran species that utilize analogous male-produced pheromones remain unclear. Thus, our goals were to test the hypotheses that 1) cerambycids respond to pheromones in a dose-dependent (= release rate-dependent) manner and 2) pheromone emission is density-dependent. If true, these characteristics of pheromone use could suggest that cerambycids utilize an optimal density strategy to limit competition for scarce and ephemeral hosts, i.e., the stressed or dying trees that typically constitute their larval hosts. Attraction of beetles to a range of release rates of two common pheromone components - 2-methylbutanol and 3-hydroxyhexan-2-one - was tested in field trials. Responses, as measured by the number of beetles caught in pheromone-baited traps, increased with release rates for five endemic species, even at the highest rates tested (~1450 µg/h for 2-methylbutanol and ~720 µg/h for 3-hydroxyhexan-2-one). The effect of density of conspecific males on per capita pheromone production was tested by collecting the volatiles produced by individuals, pairs, or groups of three or four male Phymatodes grandis Casey. Frequency of pheromone production was significantly different among the treatment densities, and emission rates of the pheromone (R)-2-methylbutanol decreased with increasing density. These results are discussed in the context of a possible optimal density strategy used by cerambycids, and more broadly, in relation to the use of male-produced aggregation-sex pheromones by other coleopterans. In addition, we report the identification of the pheromones of four of our five test species, specifically, Phymatodes obliquus Casey, Brothylus conspersus LeConte, Brothylus gemmulatus LeConte, and Xylotrechus albonotatus Casey.

Histone deacetylases in sake yeast affect fermentation characteristics.

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3Δ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3Δ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation. Abbreviations: HDAC: Histone deacetylase; HAT: histone acetyltransferase; K701: sake yeast Kyokai No. 701; PCR: polymerase chain reaction; HPLC: high performance liquid chromatography; E/A: Ester/Alcohol; BCAA: branched chain-amino acid; Atf: alcohol acetyltransferase.

Engineering Corynebacterium glutamicum Mutants for 3-Methyl-1-butanol Production.

3-Methyl-1-butanol (3MB) is a promising biofuel that can be produced from 2-ketoisocaproate via the common L-leucine biosynthesis pathway. Corynebacterium glutamicum was chosen as a host bacterium because of its strong resistance to isobutanol. In the current study, several strategies were designed to overproduce 3MB in C. glutamicum through a non-fermentation pathway. The engineered C. glutamicum mutant was obtained by silencing the pyruvate dehydrogenase gene complex (aceE) and deleting the lactic dehydrogenase gene (ldh), followed by mutagenesis with diethyl sulfate (DES) and selection with Fmoc-3-4-thiazolyl-L-alanine (FTA). The mutant could produce 659 mg/L of 3MB after 12 h of incubation. To facilitate carbon flux to 3MB biosynthesis, the engineered recombinant was also constructed without branched-chain acid aminotransferase (ilvE) activity by deleting the ilvE gene. This recombinant could produce 697 mg/L of 3MB after 12 h of incubation.

Cooperative defence operates by social modulation of biogenic amine levels in the honey bee brain.

The defence of a society often requires that some specialized members coordinate to repel a threat at personal risk. This is especially true for honey bee guards, which defend the hive and may sacrifice their lives upon stinging. Central to this cooperative defensive response is the sting alarm pheromone, which has isoamyl acetate (IAA) as its main component. Although this defensive behaviour has been well described, the neural mechanisms triggered by IAA to coordinate stinging have long remained unknown. Here we show that IAA upregulates brain levels of serotonin and dopamine, thereby increasing the likelihood of an individual bee to attack and sting. Pharmacological enhancement of the levels of both amines induces higher defensive responsiveness, while decreasing them via antagonists decreases stinging. Our results thus uncover the neural mechanism by which an alarm pheromone recruits individuals to attack and repel a threat, and suggest that the alarm pheromone of honey bees acts on their response threshold rather than as a direct trigger.

Synthetic polyprenol-pyrophosphate linked oligosaccharides are efficient substrates for mycobacterial galactan biosynthetic enzymes.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall that is critical for their survival. The largest structural component of the cell wall, the mycolyl-arabinogalactan-peptidoglycan complex, has at its core a galactan domain composed of d-galactofuranose residues. Mycobacterial galactan biosynthesis has been proposed to involve two glycosyltransferases, GlfT1 and GlfT2, which elongate polyprenol-pyrophosphate linked glycosyl acceptor substrates using UDP-galactofuranose as the donor substrate. We here report the first chemical synthesis of GlfT1 and GlfT2 acceptor substrates containing pyrophosphate and polyprenol moieties (compounds 3, 4, 22 and 23). The approach involves chemical synthesis of an oligosaccharide, subsequent phosphorylation at the reducing end and coupling to a polyprenol phosphate. These compounds were shown to be substrates for either GlfT1 (22 and 23) or GlfT2 (3 and 4) and all were substantially more active than the corresponding alkyl glycoside substrates reported previously. Mass spectrometric analysis of the products formed from the reaction of 3, 4, 22 and 23 with the respective cognate enzyme and UDP-galactofuranose provide additional evidence for the galactan biosynthetic model in which GlfT1 adds the first two galactofuranose residues with the remainder being installed via GlfT2. Overall, these results highlight the importance of the pyrophosphate motif in recognition of acceptor substrates by both enzymes and demonstrate a straightforward route for the preparation of such compounds. The work also provides additional support for the process by which this important glycan is biosynthesized using, for the first time, close structural analogs to the natural substrates.

Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli.

The biotechnological production of isoprene and isopentenol has recently been studied. Isoprene, which is currently made mainly from petroleum, is an important platform chemical for synthesizing pesticides, medicines, oil additives, fragrances, and more and is especially important in the rubber production industry. Isopentenols, which have better combustion properties than well-known biofuels (ethanol), have recently received more attention. Supplies of petroleum, the conventional source of isoprene and isopentenols, are unsustainable, and chemical synthesis processes could cause serious environmental problems. As an alternative, the biosynthesis of isoprene and isopentenols in cell factories is more sustainable and environmentally friendly. With a number of advantages over other microorganisms, Escherichia coli is considered to be a powerful workhorse organism for producing these compounds. This review will highlight the recent advances in metabolic engineering for isoprene and isopentenol production, especially using E. coli cell factories.

The history and recent advances in research of polyprenol and its derivatives.

The reduction pathway leading to the formation of dolichol was clarified in 2010 with the identification of SRD5A3, which is the polyprenol reductase. The finding inspired us to reanalyze the length of the major chain of polyprenol and dolichol from several plant leaves, including mangrove plants, as well as from animal and fish livers by 2D-TLC. Polyprenol- and dolichol-derived metabolites such as polyprenylacetone and epoxydolichol were found together with rubber-like prenol. This review focuses on analyses of polyprenol and its derivatives, including recently found epoxypolyprenol and polyprenylacetone. Attention has also been paid to the chromatographic behavior of rubber-like prenol on TLC.