Combinational biosynthesis of isoprene by engineering the MEP pathway in Escherichia coli

As an important feedstock in petrochemistry, isoprene is used in a wide range of industrial applications. It is produced almost entirely from petrochemical sources; however, these sources are being progressively depleted. A reliable biological process for isoprene production utilizing renewable feedstocks would be an industry-redefining development. There are two biosynthetic pathways producing isoprene: the mevalonate (MVA) pathway and the methyl erythritol 1-phosphate (MEP) pathway. In this study, the MEP pathway was modified in Escherichia coli BL21 (DE3) to produce isoprene. The isoprene synthase (IspS) gene chemically synthesized from Populus alba after codon optimization for expression in E. coli was heterologously expressed. The endogenous genes of 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) were over-expressed. The isopentenyl pyrophosphate isomerase (Idi) gene from Streptococcus pneumoniae was exogenously over-expressed, and farnesyl diphosphate synthase (ispA) was weakened to enhance the yield. The control strain harboring empty plasmids did not emit any isoprene. The over-expression of the DXR gene only had little impact on the yield of isoprene. Idi from S. pneumoniae played a significant role in the improvement of isoprene production. The highest yield was achieved by an ispA-weakened DXS-IDI-IspS recombinant with 19.9 mg/l isoprene, which resulted in a 33-fold enhancement of the isoprene yield from the IspS recombinant.     

Isoprene Production Via the Mevalonic Acid Pathway in Escherichia coli (Bacteria)

There is a need to develop renewable fuels and chemicals that will help meet global demands for energy and synthetic chemistry feedstock, without contributing to climate change or environmental degradation. Isoprene (C₅H₈) is one such key chemical ingredient, required for the production of synthetic rubber or plastic products, and a potential biofuel. Enabling a sustainable microbial fermentation for the production of isoprene is an attractive alternative to a petroleum origin. This work demonstrates transgenic expression of the Pueraria montana (kudzu vine) isoprene synthase gene (kIspS) and heterologous isoprene production in Escherichia coli. Enhancements in the amount of E. coli isoprene production were achieved upon over-expression of the native 2-C-methyl-d-erythritol-4-phosphate (MEP) biosynthetic pathway and, independently, upon heterologous over-expression of the entire mevalonic acid (MVA) pathway. A direct comparison of the efficiency of cellular organic carbon flux through the MEP and MVA pathways is provided, under conditions when these are expressed in the same host using the same plasmid, and same ribosome-binding sites (RBS). These alternative isoprenoid biosynthetic pathways were assembled in and expressed through a superoperon, suitable for transformation of E. coli. Introduction of specific RBS and nucleotide spacers between individual genes in the superoperon structure enabled maximal expression in E. coli batch cultures and translated to an improved production from 0.4?mg isoprene per liter of culture (control) to 5?mg isoprene per liter of culture (MEP superoperon transformants) and up to 320?mg isoprene per liter of culture (MVA superoperon transformants). This 800-fold increase in isoprene concentration from the MVA transformants and the attendant isoprene-to-biomass 0.78:1 carbon partitioning ratio suggested that the engineered MVA pathway introduces a bypass in the flux of endogenous substrate in E. coli to isopentenyl-diphosphate and dimethylallyl-diphosphate, thus overcoming flux limitations imposed upon the regulation of the native MEP pathway by the cell.     

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two‐phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway‐only control. Biotechnol. Bioeng. 2010;107: 421–429. © 2010 Wiley Periodicals, Inc.     

High titer mevalonate fermentation and its feeding as a building block for isoprenoids (isoprene and sabinene) production in engineered Escherichia coli

Isoprenoids are important fine chemicals as material monomers, advanced fuels and pharmaceuticals. A variety of natural isoprenoids can be synthesized by engineered microbial strains. This work established a process by dividing the current isoprenoid pathway into the upstream fermentation process, from sugar to mevalonate (MVA), and the downstream process, from MVA to the target isoprenoids. The results showed that significant differences existed in the process conditions between the upstream and downstream fermentations. After individually optimizing the process conditions, the upstream MVA production (84.0 g/L, 34.0% and 1.8 g/ L/h) and downstream isoprene production (11.0 g/L and 0.23 g/L/h) were greatly improved in this two-step process. Flask fermentation experiments also confirmed that two-step route can significantly improve the sabinene titer to 150 mg/L (6.5-fold of the sabinene titer in an earlier flask study of our lab). Therefore, the two-step route proposed in this study may have potential benefits towards the current isoprenoids production directly from glucose. The high titer and yield of MVA indicate that MVA has great potential to be more broadly utilized as starting precursor in synthetic biology.     

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

Branched C₅ alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C₅ alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C₅ alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete “decoupling” of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C₅ alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.     

基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

2-C-甲基-D-赤藻糖醇-4-磷酸(2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichiacoli 唯一的萜类前体合成途径,研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP 途径的调控所知非常有限,故单独强化MEP 途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP 途径基因强化E. coli 萜类合成的遗传改造策略和发酵过程补糖控制优化,尝试更有效地释放MEP 途径的潜力,建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2 基因和枯草芽胞杆菌Bacillus subtilis idi 基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2 倍。随后针对该菌株建立了高密度发酵过程,发现稳定期的中前期(24?72 h) 是产物合成的关键期,通过稳定期补糖速率的调整,明显改善了产物合成速度,使紫槐二烯的产量从2.5 g/L 提高到了4.85 g/L,但不影响产物积累的周期。考虑到72 h 后菌体老化可能会影响产物合成,进一步采取了调整对数期的补糖速率控制菌体生长的策略,使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP 途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。     

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria,using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

High-level De novo biosynthesis of arbutin in engineered Escherichia coli

Arbutin is a hydroquinone glucoside compound existing in various plants. It is widely used in pharmaceutical and cosmetic industries owing to its well-known skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. Currently, arbutin is usually produced by plant extraction or enzymatic processes, which suffer from low product yield and expensive processing cost. In this work, we established an artificial pathway in Escherichia coli for high-level production of arbutin from simple carbon sources. First, a 4-hydroxybenzoate 1-hydroxylase from Candida parapsilosis CBS604 and a glucosyltransferase from Rauvolfia serpentina were characterized by in vitro enzyme assays. Introduction of these two genes into E. coli led to the production of 54.71 mg/L of arbutin from glucose. Further redirection of carbon flux into arbutin biosynthesis pathway by enhancing shikimate pathway genes enabled production of 3.29 g/L arbutin, which is a 60-fold increase compared with the initial strain. Final optimization of glucose concentration added in the culture medium was able to further improve the titer of arbutin to 4.19 g/L in shake flasks experiments, which is around 77-fold higher than that of initial strain. This work established de novo biosynthesis of arbutin from simple carbon sources and provided a generalizable strategy for the biosynthesis of shikimate pathway derived chemicals. The high titer achieved in our engineered strain also indicates the potential for industrial scale bio-manufacturing of arbutin.     

Biosynthesis of isoprene in <Emphasis Type="Italic">Escherichia coli</Emphasis> via methylerythritol phosphate (MEP) pathway

Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-d-xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.     

Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens‐mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene‐producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene‐forming gene. Notably, by co‐overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well‐established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.     

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k_cat or K_i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.     

Combining De Ley–Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from d-galactose

An engineered Escherichia coli strain was developed for enhanced isoprene production using d-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley–Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from d-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct d-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze d-galactose conversion to d-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD–MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD–MEP pathway.     

Geranyl diphosphate synthase: An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli

The expression level of geranyl diphosphate synthase (GPPS) was suspected to play a key role for geraniol production in recombinant Escherichia coli harboring an entire mevalonate pathway operon and a geraniol synthesis operon. The expression of GPPS was optimized by using ribosomal binding sites (RBSs) designed to have different translation initiation rates (TIRs). The RBS strength in TIR window of 500 arbitrary unit (au)–1400 au for GPPS appears to be suitable for balancing the geraniol biosynthesis pathway in this study. With the TIR of 500 au, the highest production titer of geraniol was obtained at a level of 1119 mg/L, which represented a 6-fold increase in comparison with the previous titer of 183 mg/L. The TIRs of GPPS locating out of range of the optimal window (500–1400 au) caused significant decreases of cell growth and geraniol production. It was suspected to result from metabolic imbalance and plasmid instability in geraniol production by inappropriate expression level of GPP synthase. Our results collectively indicated GPPS as an important regulation point in balancing a recombinant geraniol synthesis pathway. The GPPS-based regulation approach could be applicable for optimizing microbial production of other monoterpenes.     

Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis

Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) reached 722.46 mg/L and 23.16 mg/g dry cell weight (DCW), respectively, in 5.0 L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSP_gadE-MevT_TIGR-MevB_TIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition.