CYPArm2 is a CYP450 Monooxygenase with Protoilludene 13-Hydroxylase Activity Involved in the Biosynthesis of Armillyl Orsellinate-Type Sesquiterpenoids.

The melleolides are a family of structurally and functionally diverse sesquiterpenoids with potential applications as fungicides, antimicrobials, and cancer therapeutics. The initial and terminal steps of the biosynthesis pathway in Armillaria spp. have been characterized, but the intermediate steps are unclear. Biosynthetic gene clusters in A. mellea and A. gallica were shown to encode a terpene cyclase, a polyketide synthase, and four CYP450 monooxygenases. We have characterized CYPArm3, which is responsible for the hydroxylation of Δ-6-protoilludene, but the functions of the other CYP450s remain to be determined. Here we describe CYPArm2, which accepts Δ-6-protoilludene and 8α-hydroxy-6-protoilludene as substrates. To investigate the products in more detail, we generated recombinant Saccharomyces cerevisiae strains overexpressing CYPArm2 in combination with the previously characterized protoilludene synthase and 8α-hydroxylase. Using this total biosynthesis approach, sufficient quantities of product were obtained for NMR spectroscopy. This allowed the identification of 8α,13-dihydroxy-protoilludene, confirming that CYPArm2 is a protoilludene 13-hydroxylase.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2.

BACKGROUND: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. RESULTS: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L-1 butanol and 133 mg L-1 hexanol from fructose in complex medium, and 174 mg L-1 butanol and 15 mg L-1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L-1) at the expense of butanol (158 mg L-1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L-1 butanol and 393 mg L-1 hexanol. CONCLUSIONS: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Isolation of a gene cluster from Armillaria gallica for the synthesis of armillyl orsellinate-type sesquiterpenoids.

Melleolides and armillyl orsellinates are protoilludene-type aryl esters that are synthesized exclusively by parasitic fungi of the globally distributed genus Armillaria (Agaricomycetes, Physalacriaceae). Several of these compounds show potent antimicrobial and cytotoxic activities, making them promising leads for the development of new antibiotics or drugs for the treatment of cancer. We recently cloned and characterized the Armillaria gallica gene Pro1 encoding protoilludene synthase, a sesquiterpene cyclase catalyzing the pathway-committing step to all protoilludene-type aryl esters. Fungal enzymes representing secondary metabolic pathways are sometimes encoded by gene clusters, so we hypothesized that the missing steps in the pathway to melleolides and armillyl orsellinates might be identified by cloning the genes surrounding Pro1. Here we report the isolation of an A. gallica gene cluster encoding protoilludene synthase and four cytochrome P450 monooxygenases. Heterologous expression and functional analysis resulted in the identification of protoilludene-8α-hydroxylase, which catalyzes the first committed step in the armillyl orsellinate pathway. This confirms that ∆-6-protoilludene is a precursor for the synthesis of both melleolides and armillyl orsellinates, but the two pathways already branch at the level of the first oxygenation step. Our results provide insight into the synthesis of these valuable natural products and pave the way for their production by metabolic engineering. KEY POINTS: • Protoilludene-type aryl esters are bioactive metabolites produced by Armillaria spp. • The pathway-committing step to these compounds is catalyzed by protoilludene synthase. • We characterized CYP-type enzymes in the cluster and identified novel intermediates.

Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis.

BACKGROUND: Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. RESULTS: We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium. DISCUSSION: These findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.

Structural Basis for Norovirus Inhibition by Human Milk Oligosaccharides.

Histo-blood group antigens (HBGAs) are important binding factors for norovirus infections. We show that two human milk oligosaccharides, 2'-fucosyllactose (2'FL) and 3-fucosyllactose (3FL), could block norovirus from binding to surrogate HBGA samples. We found that 2'FL and 3FL bound at the equivalent HBGA pockets on the norovirus capsid using X-ray crystallography. Our data revealed that 2'FL and 3FL structurally mimic HBGAs. These results suggest that 2'FL and 3FL might act as naturally occurring decoys in humans.

Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose.

BACKGROUND: Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such as Clostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced. We therefore developed a metabolic engineering approach in C. cellulolyticum to expand its natural product spectrum and to fine tune the engineered metabolic pathways. RESULTS: Here we report the metabolic engineering of C. cellulolyticum to produce n-butanol, a next-generation biofuel and important chemical feedstock, directly from crystalline cellulose. We introduced the CoA-dependent pathway for n-butanol synthesis from C. acetobutylicum and measured the expression of functional enzymes (using targeted proteomics) and the abundance of metabolic intermediates (by LC-MS/MS) to identify potential bottlenecks in the n-butanol biosynthesis pathway. We achieved yields of 40 and 120 mg/L n-butanol from cellobiose and crystalline cellulose, respectively, after cultivating the bacteria for 6 and 20 days. CONCLUSION: The analysis of enzyme activities and key intracellular metabolites provides a robust framework to determine the metabolic flux through heterologous pathways in C. cellulolyticum, allowing further improvements by fine tuning individual steps to improve the yields of n-butanol.

Allosteric antibody inhibition of human hepsin protease.

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.

The Restriction-Modification Systems of Clostridium carboxidivorans P7.

Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction-modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain.

Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides.

Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6-7 double bond into the 7-8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns.

Impact of different trace elements on metabolic routes during heterotrophic growth of C. ljungdahlii investigated through online measurement of the carbon dioxide transfer rate.

Synthesis gas fermentation using acetogenic clostridia is a rapidly increasing research area. It offers the possibility to produce platform chemicals from sustainable C1 carbon sources. The Wood-Ljungdahl pathway (WLP), which allows acetogens to grow autotrophically, is also active during heterotrophic growth. It acts as an electron sink and allows for the utilization of a wide variety of soluble substrates and increases ATP yields during heterotrophic growth. While glycolysis leads to CO2 evolution, WLP activity results in CO2 fixation. Thus, a reduction of net CO2 emissions during growth with sugars is an indicator of WLP activity. To study the effect of trace elements and ventilation rates on the interaction between glycolysis and the WLP, the model acetogen Clostridium ljungdahlii was cultivated in YTF medium, a complex medium generally employed for heterotrophic growth, with fructose as growth substrate. The recently reported anaRAMOS device was used for online measurement of metabolic activity, in form of CO2 evolution. The addition of multiple trace elements (iron, cobalt, manganese, zinc, nickel, copper, selenium, and tungsten) was tested, to study the interaction between glycolysis and the Wood ljungdahl pathway. While the addition of iron(II) increased growth rates and ethanol production, added nickel(II) increased WLP activity and acetate formation, reducing net CO2 production by 28%. Also, higher CO2 availability through reduced volumetric gas flow resulted in 25% reduction of CO2 evolution. These online metabolic data demonstrate that the anaRAMOS is a valuable tool in the investigation of metabolic responses i.e. to determine nutrient requirements that results in reduced CO2 production. Thereby the media composition can be optimized depending on the specific goal.

Hexanol biosynthesis from syngas by Clostridium carboxidivorans P7 - product toxicity, temperature dependence and in situ extraction.

Clostridium carboxidivorans converts syngas into industrial alcohols like hexanol, but titers may be limited by product toxicity. Investigation of IC50 at 30 °C (17.5 mM) and 37 °C (11.8 mM) revealed increased hexanol tolerance at lower temperatures. To avoid product toxicity, oleyl alcohol was added as an extraction solvent, increasing hexanol production nearly 2.5-fold to 23.9 mM (2.4 g/L) at 30 °C. This titer exceeds the concentration that is acutely toxic in the absence of a solvent, confirming the hypothesis that current hexanol production is limited by product toxicity. The solvent however had no positive effect at 37 °C. Furthermore, C. carboxidivorans cell membranes adapted to the higher temperature by incorporating more saturated fatty acids, but surprisingly not to hexanol. Corn oil and sunflower seed oil were tested as alternative, inexpensive extraction solvents. Hexanol titers were similar with all solvents, but oleyl alcohol achieved the highest extraction efficiency.

Clinical Evaluation of 16-Week Supplementation with 5HMO-Mix in Healthy-Term Human Infants to Determine Tolerability, Safety, and Effect on Growth.

Human milk oligosaccharides (HMOs) are complex sugars that occur naturally in human breast milk and provide many beneficial functions. Most formula products lack HMOs or contain only the most abundant HMO, 2'-fucosyllactose; however, benefits of HMOs come from multiple sugars. We therefore developed a mixture of five HMOs (5HMO-Mix) mimicking the natural concentrations of the top five HMOs (5.75 g/L total, comprising 52% 2'-fucosyllactose, 13% 3-fucosyllactose, 26% lacto-N-tetraose, 4% 3'-sialyllactose, and 5% 6'-sialyllactose) representing the groups of neutral, neutral-fucosylated, and sialylated HMOs. We conducted the first multicenter, randomized, controlled, parallel-group clinical study assessing the safety, tolerability, and effect on growth of formula containing the 5HMO-Mix in healthy infants. We enrolled 341 subjects aged ≤14 days; 225 were randomized into groups fed either with infant formula containing 5HMO-Mix (5HMO-Mix) or infant formula without HMOs (IF) for 4 months, with the others exclusively breastfed. There were no differences in weight, length, or head circumference gain between the two formula groups. The 5HMO-Mix was well tolerated, with 5HMO-Mix and breastfed infants producing softer stools at a higher stool frequency than the control formula group. Adverse events were equivalent in all groups. We conclude that the 5HMO-Mix at 5.75 g/L in infant formula is safe and well tolerated by healthy term infants during the first months of life.

Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures.

The production of isoprene from cellulose using recombinant Clostridium cellulolyticum strains expressing isoprene synthase.

Isoprene is an important bulk chemical which is mostly derived from fossil fuels. It is used primarily for the production of synthetic rubber. Sustainable, biotechnology-based alternatives for the production of isoprene rely on the fermentation of sugars from food and feed crops, creating an ethical dilemma due to the competition for agricultural land. This issue could be addressed by developing new approaches based on the production of isoprene from abundant renewable waste streams. Here, we describe a proof-of-principle approach for the production of isoprene from cellulosic biomass, the most abundant polymer on earth. We engineered the mesophilic prokaryote Clostridium cellulolyticum, which can degrade cellulosic biomass, to utilize the resulting glucose monomers as a feedstock for the production of isoprene. This was achieved by integrating the poplar gene encoding isoprene synthase. The presence of the enzyme was confirmed by targeted proteomics, and the accumulation of isoprene was confirmed by GC-MS/MS. We have shown for the first time that engineered C. cellulolyticum can be used as a metabolic chassis for the sustainable production of isoprene.

High-Titer De Novo Biosynthesis of the Predominant Human Milk Oligosaccharide 2'-Fucosyllactose from Sucrose in Escherichia coli.

Human milk oligosaccharides (HMOs) are unique components of human breast milk. Their large-scale production by fermentation allows infant formulas to be fortified with HMOs, but current fermentation processes require lactose as a starting material, increasing the costs, bioburden, and environmental impact of manufacturing. Here we report the development of an Escherichia coli strain that produces 2'-fucosyllactose (2'-FL), the most abundant HMO, de novo using sucrose as the sole carbon source. Strain engineering required the expression of a novel glucose-accepting galactosyltransferase, overexpression of the de novo UDP-d-galactose and GDP-l-fucose pathways, the engineering of an intracellular pool of free glucose, and overexpression of a suitable α(1,2)-fucosyltransferase. The export of 2'-FL was facilitated using a sugar efflux transporter. The final production strain achieved 2'-FL yields exceeding 60 g/L after fermentation for 84 h. This efficient strategy facilitates the lactose-independent production of HMOs by fermentation, which will improve product quality and reduce the costs of manufacturing.

A safety evaluation of mixed human milk oligosaccharides in rats.

Human milk oligosaccharides (HMOs) are indigestible carbohydrates representing the third largest fraction of solutes in human breastmilk. They provide valuable prebiotic and anti-pathogenic functions in breastfed infants, but are not yet included in most infant formula products. Recent biotechnological advances now facilitate large-scale production of HMOs, providing infant formula manufacturers with the ability to supplement their products with HMOs to mimic human breastmilk. Although the safety of individual HMOs has been confirmed in preclinical toxicological studies, the safety of HMO mixtures has not been tested. We therefore performed bacterial reverse mutation and in vitro micronucleus tests and conducted a repeated-dose oral toxicity study in rats with a mixture of five HMOs (HMO MIX I), containing 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL). HMO MIX I was not genotoxic and did not induce adverse effects in the repeated dose study. The no-observed-adverse-effect-level (NOAEL) for HMO MIX I in this study is 10% in the diet (equivalent to 5.67 g HMO MIX I/kg bw/day for males and 6.97 g HMO MIX I/kg bw/day for females). Our results provide strong evidence for the safety of HMO MIX I in infant products and general foods.

Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors.

Norovirus infections cause severe gastroenteritis in millions of people every year. Infection requires the recognition of histo-blood group antigens (HBGAs), but such interactions can be inhibited by human milk oligosaccharides (HMOs), which act as structurally-similar decoys. HMO supplements could help to prevent norovirus infections, but the industrial production of complex HMOs is challenging. Here we describe a large-scale fermentation process that yields several kilograms of lacto-N-fucopentaose I (LNFP I). The product was synthesized in Escherichia coli BL21(DE3) cells expressing a recombinant N-acetylglucosaminyltransferase, ß(1,3)galactosyltransferase and α(1,2)fucosyltransferase. Subsequent in vitro enzymatic conversion produced HBGA types A1 and B1 for norovirus inhibition assays. These carbohydrates inhibited the binding of GII.17 virus-like particles (VLPs) to type A1 and B1 trisaccharides more efficiently than simpler fucosylated HMOs, which were in turn more effective than any non-fucosylated structures. However, we found that the simpler fucosylated HMOs were more effective than complex molecules such as LNFP I when inhibiting the binding of GII.17 and GII.4 VLPs to human gastric mucins and mucins from human amniotic fluid. Our results show that complex fucosylated HMOs can be produced by large-scale fermentation and that a combination of simple and complex fucosylated structures is more likely to prevent norovirus infections.

Genome shuffling in Clostridium diolis DSM 15410 for improved 1,3-propanediol production.

Several microorganisms are known for their efficient anaerobic conversion of glycerol to 1,3-propanediol, with Clostridium diolis DSM 15410 as one of the better performers in terms of molar yield and volumetric productivity. However, this performance is still insufficient to compete with established chemical processes. Previous studies have shown that high concentrations of 1,3-propanediol, glycerol, and fermentation side products can limit the productivity of C. diolis DSM 15410. Here, we describe the use of genome shuffling for improved 1,3-propanediol fermentation by the strict anaerobe C. diolis DSM 15410. By using chemical mutagenesis, strains with superior substrate and product tolerance levels were isolated and higher product yields were obtained. These superior strains were then used for genome shuffling and selection for 1,3-propanediol and organic acid tolerance. After four rounds of genome shuffling and selection, significant improvements were observed, with one strain attaining a 1,3-propanediol volumetric yield of 85 g/liter. This result represents an 80% improvement compared to the yield from the parental wild-type strain.

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

BACKGROUND: Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways. RESULTS: We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source. CONCLUSIONS: The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production.

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7+/-0.85mg/l as well as significant amounts of geranylgeraniol (33.1+/-5.6mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.