Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.     

mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5' hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5' hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 x 10(-4) and 4 x 10(-4)%, the productivity of low-copy plasmids containing the 5'-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression.     

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.     

High-level production of lycopene in metabolically engineered E. coli

Recombinant lycopene was generated by utilizing metabolically engineered Escherichia coli with yields being dependent upon inocula state. Yields were especially low in the case of cultures harboring high-copy plasmids that were established with inocula at the stationary growth phase. On the other hand, cultures derived using low-copy plasmid, however, yielded high amounts of lycopene irrespective of inocula state. Nevertheless, it showed still an inocula dependence pattern in lycopene productivity (mg/l/h). To further increase lycopene productivity, we applied a temperature-shift culture technique (37 → 25 °C). Using this method, we effectively enhanced lycopene productivity without any problematic phenomena. As a result, we were able to increase lycopene yield by approximately 20% compared to previous culture methods. In the present study, we were able to reach a final lycopene yield up to 260 mg/l for 60 h, which corresponds to the highest titer to date for the production of lycopene in E. coli.     

A Monte Carlo simulation of plasmid replication during the bacterial division cycle

Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. (c) 1996 John Wiley & Sons, Inc.     

Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production

Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.     

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or “leaky” production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI^q gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI^q-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.     

Use of copper sulphate and Paraquat as selectable resistance markers for stable maintenance of yeast recombinant plasmids

Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) in laboratory and industrial yeast strains. The plasmid, YEpS , containing the SOD2 gene was unstable in a sod2° mutant. However when Paraquat (0.5 mM) was used as a selective agent, the plasmid was maintained in the sod2° mutant but lost in the wild-type strain. When the CUP1 gene was inserted into YEpS 1 , the resulting plasmid (YEpCuS ) was 100% stable in the sod2° mutant grown in Cu -containing medium. In the absence of Cu , the proportion of plasmid-containing cells fell to 20%. YEpS was also transformed into an industrial strain, transformants could be selected in Paraquat-containing medium but showed poor stability.     

Expression of prokaryotic 1-deoxy-D-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis

Isopentenyl diphosphate (IPP) acts as the common, five-carbon building block in the biosynthesis of all isoprenoids. The first reaction of IPP biosynthesis in Escherichia coli is the formation of 1-deoxy-D-xylulose-5-phosphate, catalysed by 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). E. coli engineered to produce lycopene, was transformed with dxps genes cloned from Bacillus subtilis and Synechocystis sp. 6803. Increases in lycopene levels were observed in strains expressing exogenous DXPS compared to controls. The recombinant strains also exhibited elevated levels of ubiquinone-8. These increases corresponded with enhanced DXP synthase activity in the recombinant E. coli strains.     

Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate

To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD(600) value of 15 for 72 h.     

Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli

Lycopene is a useful phytochemical that holds great commercial value. In our study the lycopene production pathway in E. coli originating from the precursor isopentenyl diphosphate (IPP) of the non-mevalonate pathway was reconstructed. This engineered strain of E. coli accumulated lycopene intracellularly under aerobic conditions. As a next step, the production of lycopene was enhanced through metabolic engineering methodologies. Various competing pathways at the pyruvate and acetyl-CoA nodes were inactivated to divert more carbon flux to IPP and subsequently to lycopene. It was found that the ackA-pta, nuo mutant produced a higher amount of lycopene compared to the parent strain. To further enhance lycopene production, a novel mevalonate pathway, in addition to the already existing non-mevalonate pathway, was engineered. This pathway utilizes acetyl-CoA as precursor, condensing it to form acetoacetyl-CoA and subsequently leading to formation of IPP. Upon the introduction of this new pathway, lycopene production increased by over 2-fold compared to the ackA-pta, nuo mutant strain.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids.

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

<Emphasis Type="Italic">In silico</Emphasis> analysis and experimental improvement of taxadiene heterologous biosynthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Recombinant Candida utilis for the production of biotin

Biotin is an important nutritional supplement but is difficult to manufacture effectively. Here we present a trial of biotin production using the food yeast Candida utilis. In this system, we cloned the C. utilis biotin synthase (BIO2) gene, the gene of the rate-limiting enzyme for biotin biosynthesis, and assembled it under the control of a strong promoter. A series of plasmids were constructed to direct the integration of the BIO2 gene, either high-copy integration with 18S rDNA fragment or low-copy integration with URA3 or HIS3 fragment. The BIO2 gene can be successfully integrated into the C. utilis chromosome and can drive biotin production using these plasmids. The biotin yield in this system can reach 100-fold above the endogenous level in a small-scale culture. Although the biotin production is not stable if the selection pressure is removed, this system has the potential to produce biotin-rich feed or food additives directly without the requirement of further purification.     

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.