ABSTRACT
Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.
Subject(s)
Biofuels/analysis , Sustainable Development , Biosynthetic Pathways , Carbon Cycle , Humans , Lignin/metabolism , Waste ProductsABSTRACT
Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our â¼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.
Subject(s)
Polyketide Synthases , Polyketides , Polyketide Synthases/metabolism , Acyltransferases/chemistry , Catalytic Domain , Polyketides/metabolism , Substrate SpecificityABSTRACT
Cyanobacteria are promising as a biotechnological platform for production of various industrially relevant compounds, including aromatic amino acids and their derivatives, phenylpropanoids. In this study, we have generated phenylalanine resistant mutant strains (PRMs) of the unicellular cyanobacterium Synechocystis sp. PCC 6803, by laboratory evolution under the selective pressure of phenylalanine, which inhibits the growth of wild type Synechocystis. The new strains of Synechocystis were tested for their ability to secrete phenylalanine in the growth medium during cultivation in shake flasks as well as in a high-density cultivation (HDC) system. All PRM strains secreted phenylalanine into the culture medium, with one of the mutants, PRM8, demonstrating the highest specific production of 24.9 ± 7 mg L-1·OD750-1 or 610 ± 196 mg L-1 phenylalanine after four days of growth in HDC. We further overexpressed phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) in the mutant strains in order to determine the potential of PRMs for production of trans-cinnamic acid (tCA) and para-coumaric acid (pCou), the first intermediates of the plant phenylpropanoid pathway. Productivities of these compounds were found to be lower in the PRMs compared to respective control strains, except for PRM8 under HDC conditions. The PRM8 background strain in combination with PAL or TAL expression demonstrated a specific production of 52.7 ± 15 mg L-1·OD750-1tCA and 47.1 ± 7 mg L-1·OD750-1pCou, respectively, with a volumetric titer reaching above 1 g L-1 for both products after four days of HDC cultivation. The genomes of PRMs were sequenced in order to identify which mutations caused the phenotype. Interestingly, all of the PRMs contained at least one mutation in their ccmA gene, which encodes DAHP synthase, the first enzyme of the pathway for aromatic amino acids biosynthesis. Altogether, we demonstrate that the combination of laboratory-evolved mutants and targeted metabolic engineering can be a powerful tool in cyanobacterial strain development.
Subject(s)
Synechocystis , Synechocystis/genetics , Synechocystis/metabolism , Coumaric Acids , Phenylalanine/genetics , Phenylalanine/metabolismABSTRACT
Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.
Subject(s)
Escherichia coli Proteins , Mevalonic Acid , Biofuels , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Metabolic Engineering , PyrophosphatasesABSTRACT
Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8â¯mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.
Subject(s)
Hemiterpenes/biosynthesis , Metabolic Engineering , Microorganisms, Genetically-Modified , Synechocystis , Alkyl and Aryl Transferases/biosynthesis , Alkyl and Aryl Transferases/genetics , Butadienes , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics , Pueraria/enzymology , Pueraria/genetics , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO2 and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65⯵mol photonsâ¯m-2 s-1, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink.
Subject(s)
Biofuels , Biomass , Ethanol/metabolism , Metabolic Engineering , Microorganisms, Genetically-Modified , Synechocystis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/growth & development , Synechocystis/genetics , Synechocystis/growth & developmentABSTRACT
Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.
Subject(s)
Polyketide Synthases , Polyketide Synthases/metabolism , Amino Acid SequenceABSTRACT
Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of squalene from CO2. In this organism, squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a squalene titer of 5.1 âmg/L of culture was reached.
ABSTRACT
Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3 mg L-1 OD750-1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L-1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.
ABSTRACT
For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni2+ and Co2+, expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications.
Subject(s)
Biotechnology , Promoter Regions, Genetic , Ribosomes/metabolism , Synechocystis/metabolism , Bacterial Proteins/genetics , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Ethanol/metabolism , Genes, Bacterial , Light , Luminescent Proteins/genetics , Synechocystis/geneticsABSTRACT
Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.
Subject(s)
Diterpenes/metabolism , Glucosyltransferases , Metabolic Engineering , Plant Proteins , Plectranthus/genetics , Synechocystis , Glucosyltransferases/biosynthesis , Glucosyltransferases/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plectranthus/enzymology , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.