Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.     

Physicochemical and immunochemical characterization of salicylate 5-hydroxylase,m-hydroxybenzoate 6-hydroxylase and p-hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis

Salicylate 5-hydroxylase (SAL5H), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) from Gram-positive Rhodococcus erythropolis strain S1 were characterized physicochemically and immunochemically. The subunit size and amino acid composition of SAL5H, MHB6H, and PHB3H from strain S1 showed properties similar to those of other flavin-containing aromatic compound monooxygenases such as p-hydroxybenzoate hydroxylase and salicylate 1-hydroxylase (SAL1H), belonging to p-hydroxybenzoate hydroxylase-class, except for homotetrameric structure and cofactor specficity. The N-terminal amino acid sequence of MHB6H from strain S1 indicated significant similarity of ADP-binding region in the N-terminal portion of the enzyme with that known for SAL1H from Pseudomonas putida. Immunochemical properties, determined while conducting serological experiments, showed SAL5H and MHB6H from strain S1 to be immunologically different from PHB3H from strain S1, while SAL5H and MHB6H to apparently share partial antigenic determinants.     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.     

Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.     

Titer improvement of iso-migrastatin in selected heterologous <Emphasis Type="Italic">Streptomyces</Emphasis> hosts and related analysis of mRNA expression by quantitative RT–PCR

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO₃ to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.     

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-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowia lipolytica

The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non-economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, β-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the β-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.     

Salinomycin biosynthesis reversely regulates the β-oxidation pathway in Streptomyces albus by carrying a 3-hydroxyacyl-CoA dehydrogenase gene in its biosynthetic gene cluster

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non-ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil-preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl-CoA precursors for salinomycin biosynthesis with the aid of its enhanced β-oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3-hydroxyacyl-CoA dehydrogenase (FadB3), which is the third enzyme of β-oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the β-oxidation pathway rather than ethylmalonyl-CoA biosynthesis and played a very important role in regulating the rate of β-oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the β-oxidation pathway by carrying β-oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.     

Biotechnology for the production of plant secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.     

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides.     

Metabolic engineering of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> for malonyl-CoA biosynthesis to enhance heterologous production of polyketides

Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.     

Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant,antidiabetic, and sweet tasting properties

Dihydrochalcones are plant secondary metabolites comprising molecules of significant commercial interest as antioxidants, antidiabetics, or sweeteners. To date, their heterologous biosynthesis in microorganisms has been achieved only by precursor feeding or as minor by-products in strains engineered for flavonoid production. Here, the native ScTSC13 was overexpressed in Saccharomyces cerevisiae to increase its side activity in reducing p-coumaroyl-CoA to p-dihydrocoumaroyl-CoA. De novo production of phloretin, the first committed dihydrochalcone, was achieved by co-expression of additional relevant pathway enzymes. Naringenin, a major by-product of the initial pathway, was practically eliminated by using a chalcone synthase from barley with unexpected substrate specificity. By further extension of the pathway from phloretin with decorating enzymes with known specificities for dihydrochalcones, and by exploiting substrate flexibility of enzymes involved in flavonoid biosynthesis, de novo production of the antioxidant molecule nothofagin, the antidiabetic molecule phlorizin, the sweet molecule naringin dihydrochalcone, and 3-hydroxyphloretin was achieved.     

Engineering <Emphasis Type="Italic">Bacillus subtilis</Emphasis> for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression

In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P₄₃ was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.     

Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae

Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well‐known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase‐like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over‐expression of a truncated 3‐hydroxyl‐3‐methylglutaryl‐CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over‐expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over‐expressing ERG20‐BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over‐expression of tHMGR‐upc2.1 and ERG20‐BTS1‐SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed‐batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Biotechnol. Bioeng. 2012; 109: 2845–2853. © 2012 Wiley Periodicals, Inc.     

Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition

Capture and conversion of CO₂ to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry‐relevant chemicals from CO₂. However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO₂ to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP‐driven malonyl‐CoA synthesis pathway and heterologous phosphoketolase (PHK)‐phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl‐CoA directly from CO₂ in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl‐CoA‐derived biochemicals.