Alkane production from fatty alcohols by the combined reactions catalyzed by an alcohol dehydrogenase and an aldehyde-deformylating oxygenase.

PsADH, an alcohol dehydrogenase originating in Pantoea sp. was characterized and found to convert a broad variety of fatty alcohols into their corresponding aldehydes, the substrates of alkane biosynthesis. By coupling PsADH with NpAD, a cyanobacterial aldehyde-deformylating oxygenase, and by optimizing the conditions of the enzyme-catalyzed reactions, we achieved a 52% conversion of 1-tetradecanol to tridecane. We further applied this system to generate alkanes ranging from C5-17. These alkanes can be used as biofuels, suggesting that introducing a suitable alcohol dehydrogenase is an effective strategy to utilize fatty alcohols for alkane production.

Utilizing Alcohol for Alkane Biosynthesis by Introducing a Fatty Alcohol Dehydrogenase.

Alkanes produced by microorganisms are expected to be an alternative to fossil fuels as an energy source. Microbial synthesis of alkanes involves the formation of fatty aldehydes via fatty acyl coenzyme A (acyl-CoA) intermediates derived from fatty acid metabolism, followed by aldehyde decarbonylation to generate alkanes. Advancements in metabolic engineering have enabled the construction of such pathways in various microorganisms, including Escherichia coli. However, endogenous aldehyde reductases in the host microorganisms are highly active in converting fatty aldehydes to fatty alcohols, limiting the substrate pool for alkane production. To reuse the alcohol by-product, a screening of fatty alcohol-assimilating microorganisms was conducted, and a bacterial strain, Pantoea sp. strain 7-4, was found to convert 1-tetradecanol to tetradecanal. From this strain, an alcohol dehydrogenase, PsADH, was purified and found to be involved in 1-tetradecanol-oxidizing reaction. Subsequent heterologous expression of the PsADH gene in E. coli was conducted, and recombinant PsADH was purified for a series of biochemical characterizations, including cofactors, optimal reaction conditions, and kinetic parameters. Furthermore, direct alkane production from alcohol was achieved in E. coli by coexpressing PsADH with a cyanobacterial aldehyde-deformylating oxygenase and a reducing system, including ferredoxin and ferredoxin reductase, from Nostoc punctiforme PCC73102. The alcohol-aldehyde-alkane synthetic route established in this study will provide a new approach to utilizing fatty alcohols for the production of alkane biofuel. IMPORTANCE Alcohol dehydrogenases are a group of enzymes found in many organisms. Unfortunately, studies on these enzymes mainly focus on their activities toward short-chain alcohols. In this study, we discovered an alcohol dehydrogenase, PsADH, from the bacterium Pantoea sp. 7-4, which can oxidize 1-tetradecanol to tetradecanal. The medium-chain aldehyde products generated by this enzyme can serve as the substrate of aldehyde-deformylating oxygenase to produce alkanes. The enzyme found in this study can be applied to the biosynthetic pathway involving the formation of medium-chain aldehydes to produce alkanes and other valuable compounds.

Cloning of a novel gene involved in alkane biosynthesis from Klebsiella sp.

Aliphatic medium-chain alkanes, a major component of gasoline, diesel, and jet fuels, are drop-in compatible fuels. Microorganisms with the capacity to produce medium-chain alkanes are promising for the bio-production of drop-in fuel. We found that Klebsiella sp. NBRC100048 has the ability to produce medium-chain alkanes from medium-chain aldehydes. We cloned a gene involved in conversion of aldehydes to alkanes by using a genomic fosmid library derived from Klebsiella sp. NBRC100048. The gene termed orf2991 encodes 506 amino acids and shows 62% sequence homology to the aldehyde dehydrogenase of Escherichia coli, aldB. The finding of orf2991 as a novel alkane-synthesizing enzyme gene similar to E. coli aldehyde dehydrogenase family, which is generally known to catalyze a reaction oxidizing aldehydes to fatty acids, indicated a novel function of aldehyde dehydrogenase. This finding is not only significant academically but allows developing the novel manufacturing methods of alkanes fermentation.

A search for microorganisms producing medium-chain alkanes from aldehydes.

Microorganisms with medium-chain alkane-producing activity are promising for the bio-production of drop-in fuel. In this study, we screened for microorganisms producing tridecane from tetradecanal. The activity of aldehyde decarbonylation was found in a wide range of microbes. In particular, the genus Klebsiella in the Enterobacteriaceae family was found to have a high ability to produce alkanes from aldehydes via enzyme catalyzed reaction.

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae.

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

Alkaline pH enhances farnesol production by Saccharomyces cerevisiae.

External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l.

Prenyl alcohol production by expression of exogenous isopentenyl diphosphate isomerase and farnesyl diphosphate synthase genes in Escherichia coli.

Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 microg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 microg/l).

Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols.

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.

Various oils and detergents enhance the microbial production of farnesol and related prenyl alcohols.

The object of this research was improvement of prenyl alcohol production with squalene synthase-deficient mutant Saccharomyces cerevisiae ATCC 64031. On screening of many kinds of additives, we found that oils and detergents significantly enhanced the extracellular production of prenyl alcohols. Soybean oil showed the most prominent effect among the additives tested. Its effect was accelerated by a high concentration of glucose in the medium. The combination of these cultivation conditions led to the production of more than 28 mg/l of farnesol in the soluble fraction of the broth. The addition of these compounds to the medium was an effective method for large-scale production of prenyl alcohols with microorganisms.

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.