Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-d-xylulose 5-phosphate synthase (dxs), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg?g^?1?h^?1 (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.     

Amplification of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q10 production in recombinant Escherichia coli

For the enhancement of coenzyme Q₁₀ (CoQ₁₀) production, 1-deoxy-d-xylulose 5-phosphate (DXP) synthase of Pseudomonas aeruginosa was constitutively coexpressed in a recombinant Escherichia coli strain, which harbors the ddsA gene from Gluconobacter suboxydans encoding decaprenyl diphosphate synthase. It was found that the expression of the ddsA gene caused depletion of the isopentenyl diphosphate (IPP) pool in E. coli. Amplification of DXP synthase level by installing P. aeruginosa DXP synthase restored the diminished IPP pool and concomitantly resulted in approximately a twofold increase in relative content and productivity of CoQ₁₀. Maximum CoQ₁₀ concentration of 46.1 mg l⁻¹ was achieved from glucose-limited fed-batch cultivation of the recombinant E. coli strain simultaneously harboring the ddsA and dxs genes.     

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.     

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO₂. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C₅ sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.     

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.     

Functional analysis of genes involved in the biosynthesis of isoprene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.     

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.     

A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase

High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution in Escherichia coli (E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.

     

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.     

苏云金芽胞杆菌dxr1基因的转录受SigH控制

【目的】萜类化合物广泛分布在生物界,是重要的生命物质。目前发现有两条萜类化合物的生物合成途径,即甲羟戊酸(MVA)途径和2-甲基-D-赤藓糖醇-4-磷酸(MEP)途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶(DXR,EC1.1.1.267)催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶,而在苏云金芽胞杆菌(Bacillusthuringiensis,Bt)中有2个基因(dxr1和dxr2)编码DXR酶。通过分析BtHD73菌株的dxr1基因的转录活性和dxr1突变体表型,明确dxr1基因的转录调控机制和功能。【方法】通过5?RACE分析dxr1的转录起始位点;β-半乳糖苷酶活性测定分析dxr1基因启动子(Pdxr1)的转录活性;采用同源重组技术分别敲除BtHD73菌株的dxr1和dxr2基因;利用总蛋白定量确定Cry1Ac蛋白产量;利用DXR检测试剂盒检测Bt菌株的DXR活性。【结果】dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基;与出发菌株HD73相比,Pdxr1在sig H突变体中的转录活性明显降低;dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响,但使DXR活性下降。【结论】Bt中dxr1基因的转录受Sig H控制,dxr1基因的缺失影响DXR的活性。     

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and -carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1–3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.     

Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with <Emphasis Type="Italic">Escherichia coli</Emphasis>

Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.     

Molecular characterization and expression of 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose 5-phosphate reductoisomerase (DXR) gene from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.     

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Positioning Bacillus subtilis as terpenoid cell factory

Increasing demands for bioactive compounds have motivated researchers to employ micro-organisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up-to-date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and the engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as along with promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoid cell factory development.     

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.