Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions.

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Dual Regulation of Cytoplasm and Peroxisomes for Improved Α-Farnesene Production in Recombinant Pichia pastoris.

Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.

Inhibition of microbial production of the malodorous substance isovaleric acid by 4,4' dichloro 2-hydroxydiphenyl ether (DCPP).

Human body malodour is a complex phenomenon. Several types of sweat glands produce odorless secretions that are metabolized by a consortium of skin-resident microorganisms to a diverse set of malodorous substances. Isovaleric acid, a sweaty-smelling compound, is one major malodorous component produced by staphylococci with the skin-derived amino acid L-leucine as a substrate. During wearing, fabrics are contaminated with sweat and microorganisms and high humidity propagates growth and microbial malodour production. Incomplete removal of sweat residues and microorganisms from fabrics during laundry with bleach-free detergents and at low temperatures elevate the problem of textile malodour. This study aimed to analyze the inhibitory effect of the antimicrobial 4,4' dichloro 2-hydroxydiphenyl ether (DCPP) on the formation of isovaleric acid on fabrics. Therefore, GC-FID- and GC-MS-based methods for the analysis of isovaleric acid in an artificial human sweat-mimicking medium and in textile extracts were established. Here, we show that antimicrobials capable to deposit on fabrics during laundry, such as DCPP, are effective in growth inhibition of typical malodour-generating bacteria and prevent the staphylococcal formation of isovaleric acid on fabrics in a simple experimental setup. This can contribute to increased hygiene for mild laundry care approaches, where bacterial contamination and malodour production represent a considerable consumer problem.

C/N Ratio and Specific Growth Rate Plays Important Role on Enhancing Isoprene Production in Recombinant Escherichia coli.

Effect of fermentation parameters such as C/N ratio, specific growth rate, phosphate limitation, and plasmid instability on enhancing isoprene production is the focus of the current study. Isoprene productivity in the recombinant Escherichia coli K12_MVA strain showed a bell-shaped relationship with specific growth rate in bioreactor studies with isoprene volumetric productivity peaking at 0.35/h. This behavior was depicted by a production inhibition kinetic model which envisaged a serious competition between the cellular growth, acetic acid production, and isoprene biosynthesis. The model equation derived showed a reasonable fit with the experimental values. Judicious control of the growth rates and acetate accumulation by optimizing C/N ratio, phosphate concentration, and intermittent feeding strategy resulted in maximizing the carbon flux towards isoprene. Plasmid instability caused by metabolic burden posed by the presence of dual plasmids on the bacteria was simulated using first-order degradation kinetics. The experimental plasmid loss trend was in accordance with the model simulated trend, where higher plasmid loss correlated with higher specific growth rates. Modulating the growth rate, acetate accumulation, and plasmid instability resulted in achieving maximum isoprene volumetric productivity of 1.125 g/l/h with 46.67% of carbon flux towards isoprene and a isoprene titre of 18 g/l in 16 h fermentation run.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Anaerobic Production of Isoprene by Engineered Methanosarcina Species Archaea.

Isoprene is a valuable petrochemical used for a wide variety of consumer goods, such as adhesives and synthetic rubber. We were able to achieve a high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina species methanogens, it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show that engineered methanogens direct up to 4% of total carbon flux to isoprene, demonstrating that methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria, and from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate that isoprene synthesis depletes membrane precursors and redirects electron flux, enabling isoprene to be a major metabolic product. Our results demonstrate that methanogens are a promising engineering chassis for renewable isoprene synthesis.IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs relative to those for petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The "isoprenogen" strains we developed have potential to be used for industrial production of inexpensive renewable isoprene.

PATHcre8: A Tool That Facilitates the Searching for Heterologous Biosynthetic Routes.

Developing computational tools that can facilitate the rational design of cell factories producing desired products at increased yields is challenging, as the tool needs to take into account that the preferred host organism usually has compounds that are consumed by competing reactions that reduce the yield of the desired product. On the other hand, the preferred host organisms may not have the native metabolic reactions needed to produce the compound of interest; thus, the computational tool needs to identify the metabolic reactions that will most efficiently produce the desired product. In this regard, we developed the generic tool PATHcre8 to facilitate an optimized search for heterologous biosynthetic pathway routes. PATHcre8 finds and ranks biosynthesis routes in a large number of organisms, including Cyanobacteria. The tool ranks the pathways based on feature scores that reflect reaction thermodynamics, the potentially toxic products in the pathway (compound toxicity), intermediate products in the pathway consumed by competing reactions (product consumption), and host-specific information such as enzyme copy number. A comparison with several other similar tools shows that PATHcre8 is more efficient in ranking functional pathways. To illustrate the effectiveness of PATHcre8, we further provide case studies focused on isoprene production and the biodegradation of cocaine. PATHcre8 is free for academic and nonprofit users and can be accessed at https://www.cbrc.kaust.edu.sa/pathcre8/.

Biosynthesis of Biscognienyne†B Involving a Cytochrome P450-Dependent Alkynylation.

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.

Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

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 productivity in hybrid-poplar plantations without isoprene emission to the atmosphere.

Hybrid-poplar tree plantations provide a source for biofuel and biomass, but they also increase forest isoprene emissions. The consequences of increased isoprene emissions include higher rates of tropospheric ozone production, increases in the lifetime of methane, and increases in atmospheric aerosol production, all of which affect the global energy budget and/or lead to the degradation of air quality. Using RNA interference (RNAi) to suppress isoprene emission, we show that this trait, which is thought to be required for the tolerance of abiotic stress, is not required for high rates of photosynthesis and woody biomass production in the agroforest plantation environment, even in areas with high levels of climatic stress. Biomass production over 4 y in plantations in Arizona and Oregon was similar among genetic lines that emitted or did not emit significant amounts of isoprene. Lines that had substantially reduced isoprene emission rates also showed decreases in flavonol pigments, which reduce oxidative damage during extremes of abiotic stress, a pattern that would be expected to amplify metabolic dysfunction in the absence of isoprene production in stress-prone climate regimes. However, compensatory increases in the expression of other proteomic components, especially those associated with the production of protective compounds, such as carotenoids and terpenoids, and the fact that most biomass is produced prior to the hottest and driest part of the growing season explain the observed pattern of high biomass production with low isoprene emission. Our results show that it is possible to reduce the deleterious influences of isoprene on the atmosphere, while sustaining woody biomass production in temperate agroforest plantations.

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis.

AIMS: Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. METHODS AND RESULTS: We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5 g l-1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. CONCLUSIONS: This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

Production of Bio-Based Isoprene by the Mevalonate Pathway Cassette in Ralstonia eutropha.

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter (1.9 ± 0.24 µg/l) than when using the lac promoter (1.5 ± 0.2 µg/l). Additionally, the use of three J5 promoters was more efficient (3.8 ± 0.18 µg/l) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.

Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass Taraxacum Kok-saghyz.

The rubber grass Taraxacum kok-saghyz (TKS) contains large amounts of natural rubber (cis-1,4-polyisoprene) in its enlarged roots and it is an alternative crop source of natural rubber. Natural rubber biosynthesis (NRB) and storage in the mature roots of TKS is a cascade process involving many genes, proteins and their cofactors. The TKS genome has just been annotated and many NRB-related genes have been determined. However, there is limited knowledge about the protein regulation mechanism for NRB in TKS roots. We identified 371 protein species from the mature roots of TKS by combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Meanwhile, a large-scale shotgun analysis of proteins in TKS roots at the enlargement stage was performed, and 3545 individual proteins were determined. Subsequently, all identified proteins from 2-DE gel and shotgun MS in TKS roots were subject to gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and most proteins were involved in carbon metabolic process with catalytic activity in membrane-bounded organelles, followed by proteins with binding ability, transportation and phenylpropanoid biosynthesis activities. Fifty-eight NRB-related proteins, including eight small rubber particle protein (SRPP) and two rubber elongation factor(REF) members, were identified from the TKS roots, and these proteins were involved in both mevalonate acid (MVA) and methylerythritol phosphate (MEP) pathways. To our best knowledge, it is the first high-resolution draft proteome map of the mature TKS roots. Our proteomics of TKS roots revealed both MVA and MEP pathways are important for NRB, and SRPP might be more important than REF for NRB in TKS roots. These findings would not only deepen our understanding of the TKS root proteome, but also provide new evidence on the roles of these NRB-related proteins in the mature TKS roots.

Enhancing the synthesis of latex clearing protein by different cultivation strategies.

In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1VH2), a key enzyme for the initial cleavage of the rubber backbone. Cultivations using a recombinant strain of Escherichia coli were optimized to overcome poor solubility of Lcp1VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1VH2. A specific maximum production rate of 28.3 mg Lcp1VH2 g-1 cell dry weight h-1 was obtained at 25 °C and at agitation rates between 200-300 rpm. The activity of Lcp1VH2 was strongly influenced by variations in the cultivation temperature with a specific maximum activity of 0.81 U mg-1 in cultures incubated at 30 °C. Besides cultivation-based optimization, also the strategy of fusion protein expression with NusA was successfully applied. The in vivo solubility of the Lcp1VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1VH2. The fusion protein of Lcp1VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.

Improving the production of isoprene and 1,3-propanediol by metabolically engineered Escherichia coli through recycling redox cofactor between the dual pathways.

The biosynthesis of isoprene by microorganisms is a promising green route. However, the yield of isoprene is limited due to the generation of excess NAD(P)H via the mevalonate (MVA) pathway, which converts more glucose into CO2 or undesired reduced by-products. The production of 1,3-propanediol (1,3-PDO) from glycerol is a typical NAD(P)H-consuming process, which restricts 1,3-PDO yield to ~ 0.7 mol/mol. In this study, we propose a strategy of redox cofactor balance by coupling the production of isoprene with 1,3-PDO fermentation. With the introduction and optimization of the dual pathways in an engineered Escherichia coli, ~ 85.2% of the excess NADPH from isoprene pathway was recycled for 1,3-PDO production. The best strain G05 simultaneously produced 665.2 mg/L isoprene and 2532.1 mg/L 1,3-PDO under flask fermentation conditions. The yields were 0.3 mol/mol glucose and 1.0 mol/mol glycerol, respectively, showing 3.3- and 4.3-fold improvements relative to either pathway independently. Since isoprene is a volatile organic compound (VOC) whereas 1,3-PDO is separated from the fermentation broth, their coproduction process does not increase the complexity or cost for the separation from each other. Hence, the presented strategy will be especially useful for developing efficient biocatalysts for other biofuels and biochemicals, which are driven by cofactor concentrations.

Isoprene Acts as a Signaling Molecule in Gene Networks Important for Stress Responses and Plant Growth.

Isoprene synthase converts dimethylallyl diphosphate to isoprene and appears to be necessary and sufficient to allow plants to emit isoprene at significant rates. Isoprene can protect plants from abiotic stress but is not produced naturally by all plants; for example, Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) do not produce isoprene. It is typically present at very low concentrations, suggesting a role as a signaling molecule; however, its exact physiological role and mechanism of action are not fully understood. We transformed Arabidopsis with a Eucalyptus globulus isoprene synthase The regulatory mechanisms of photosynthesis and isoprene emission were similar to those of native emitters, indicating that regulation of isoprene emission is not specific to isoprene-emitting species. Leaf chlorophyll and carotenoid contents were enhanced by isoprene, which also had a marked positive effect on hypocotyl, cotyledon, leaf, and inflorescence growth in Arabidopsis. By contrast, leaf and stem growth was reduced in tobacco engineered to emit isoprene. Expression of genes belonging to signaling networks or associated with specific growth regulators (e.g. gibberellic acid that promotes growth and jasmonic acid that promotes defense) and genes that lead to stress tolerance was altered by isoprene emission. Isoprene likely executes its effects on growth and stress tolerance through direct regulation of gene expression. Enhancement of jasmonic acid-mediated defense signaling by isoprene may trigger a growth-defense tradeoff leading to variations in the growth response. Our data support a role for isoprene as a signaling molecule.

Improvement of isoprene production in Escherichia coli by rational optimization of RBSs and key enzymes screening.

BACKGROUND: As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes. RESULTS: Firstly, screening of key enzymes, IDI, MK and IspS, from other organisms and then RBS optimization of the key enzymes were carried out. The strain utilized IDIsa was firstly detected to produce more isoprene than other IDIs. IDIsa expression was improved after RBS modification, leading to 1610-fold increase of isoprene production. Secondly, RBS sequence optimization was performed to reduce translation initiation rate value of non-key enzymes, ERG19 and MvaE. Decreased ERG19 and MvaE expression and increased isoprene production were detected. The final strain showed 2.6-fold increase in isoprene production relative to the original strain. Furthermore, for the first time, increased key enzyme expression and decreased non-key enzyme expression after RBS sequence optimization were obviously detected through SDS-PAGE analysis. CONCLUSIONS: This study prove that desired enzyme expression and increased isoprene production were obtained after RBS sequence optimization. RBS optimization of genes could be a powerful strategy for metabolic engineering of strain. Moreover, to increase the production of engineered strain, attention should not only be focused on the key enzymes, but also on the non-key enzymes.

Multi-modular engineering for renewable production of isoprene via mevalonate pathway in Escherichia coli.

AIMS: To establish the biotechnology platforms for production of bio-based chemicals in various micro-organisms is considered as a promising target to improve renewable production of isoprene. METHODS AND RESULTS: In this study, we heterologously expressed the mevalonate (MVA) isoprene biosynthesis pathway, and explored three strategies of increasing isoprene production in Escherichia coli. We first manipulated the expression levels of the MVA pathway genes through changing the gene cassettes and promoters. To introduce cofactor engineering, we then overexpressed NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene from Clostridium acetobutylicum to supply available NADPH. To reduce the inhibitory by-product accumulation, we finally knocked out acetate-producing genes, phosphate acetyl transferase and pyruvate oxidase B in E. coliJM109 (DE3), decreasing acetate accumulation 89% and increasing isoprene production 39%. The strategies described here finally increased the isoprene titre to 92 mg l-1 in two-gene deletion strain JMAB-4T7P1Trc, increasing 2·6-fold comparing to strain JM7T7. CONCLUSION: The multimodularly engineering approaches including promoter engineering, cofactor engineering and by-product reducing could be used to improve isoprene production in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic strategies in this study show us directions for further studies to promote transformation of renewable sources to isoprene.

Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production.

Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47 mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.