Lycopene Biosynthesis Pathway Research Articles

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway.

Lycopene is a common carotenoid found mainly in ripe red fruits and vegetables that is widely used in the food industry due to its characteristic color and health benefits. Microbial synthesis of lycopene is gradually replacing the traditional methods of plant extraction and chemical synthesis as a more economical and productive manufacturing strategy. The biosynthesis of lycopene is a typical multienzyme cascade reaction, and it is important to understand the characteristics of each key enzyme involved and how they are regulated. In this paper, the catalytic characteristics of the key enzymes involved in the lycopene biosynthesis pathway and related studies are first discussed in detail. Then, the strategies applied to the key enzymes of lycopene synthesis, including fusion proteins, enzyme screening, combinatorial engineering, CRISPR/Cas9-based gene editing, DNA assembly, and scaffolding technologies are purposefully illustrated and compared in terms of both traditional and emerging multienzyme regulatory strategies. Finally, future developments and regulatory options for multienzyme synthesis of lycopene and similar secondary metabolites are also discussed.

Identification of genome integration sites for developing a CRISPR-based gene expression toolkit in Yarrowia lipolytica.

SummaryWith the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR‐based gene expression toolkit of Y. lipolytica. By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR‐based gene expression toolkit developed in this study will facilitate the genetic engineering of Y. lipolytica.

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene.

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

Design and tailoring of an artificial DNA scaffolding system for efficient lycopene synthesis using zinc-finger-guided assembly.

A highly efficient lycopene production system was constructed by assembling enzymes fused to zinc-finger motifs on DNA scaffolds in vitro and in vivo. Three key enzymes of the lycopene synthesis pathway, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, were fused with zinc-finger proteins, expressed and purified. Recombinant plasmids of the pS series containing DNA scaffolds that the zinc-finger proteins can specifically bind to were constructed. In the in vitro system, the production efficiency of lycopene was improved greatly after the addition of the scaffold plasmid pS231. Subsequently, the plasmid pET-AEBI was constructed and introduced into recombinant Escherichia coli BL21 (DE3) for expression, together with plasmids of the pS series. The lycopene production rate and content of the recombinant strain pp231 were higher than that of all strains carrying the DNA scaffold and the control. With the addition of cofactors and substrates in the lycopene biosynthesis pathway, the lycopene yield of pp231 reached 632.49mg/L at 40h, representing a 4.7-fold increase compared to the original recombinant strain pA1A3. This DNA scaffold system can be used as a platform for the construction and production of many biochemicals synthesized via multi-enzyme cascade reactions.

Yeast Synthetic Terminators: Fine Regulation of Strength through Linker Sequences.

The design of improved synthetic components is an important research field in synthetic biology. The terminator, responsible for terminating gene transcription, is a necessary component for yeast gene expression. The efficiency element, the positioning element and the poly(A) site have been identified as the constituent parts necessary for the yeast terminator to perform its function. However, the functions of linker 1 (situated between the efficiency element and the positioning element) and linker 2 [between the positioning element and the poly(A) site] in the terminator are still controversial. Here, we have thus designed and synthesized a yeast synthetic terminator library incorporating random 10 bp linker 1 units. For indirect characterization of the strengths of 266 synthetic terminators with the aid of the enhanced green fluorescent protein (eGFP), their fluorescence intensity (FI) values were determined; they ranged from 2.3648 to 3.5270, thus indicating that the strength of yeast terminator can be finely adjusted by changing the linker 1 sequence. The strength increased with decreasing GC content in linker 1, with a T-rich linker 1 helping to enhance terminator strength further. Reducing the stem length can increase the gene expression in cases of weak and medium-strength terminators but decreases the gene expression of strong terminators. Deletion of linker 2 seems to have a positive effect on weak and medium-strength terminators. Construction of a lycopene biosynthesis pathway with synthetic terminators effectively regulated lycopene synthesis, thus indicating that it is highly feasible to use terminators for fine regulation of gene and pathway expression.

Exploring natural biodiversity to expand access to microbial terpene synthesis

BackgroundTerpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established—mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes.ResultsThe potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule.ConclusionIPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.

Improving lycopene production in Saccharomyces cerevisiae through optimizing pathway and chassis metabolism

Lycopene, a tetraterpenoid containing 13 double bonds, has important pharmaceutical and industrial application values. However, unstable supply of raw for extracted from natural host and residue of chemical reagents in chemically synthesized products have limited the quality of lycopene. Engineering microorganism is thus regarded as an alternative and attractive route for lycopene production. To improve the ability of engineered yeast to synthesize lycopene, here we have optimized the lycopene biosynthesis pathway and chassis metabolism. The results have verified that S. cerevisiae CEN.PK2-1C was more suitable for lycopene production because of its strong ability of synthesizing precursors of terpenoids. Compared to use inducible promoter, the use of constitutive promoter controlling pathway expression was more efficient for balancing lycopene synthesis and chassis metabolism. GGPP was identified the rate-controlling metabolite. Through expanding GGPP pool and MVA pathway, lycopene production was thus significantly increased. By citric acid fed-batch fermentation, the yield of lycopene finally reached 115.64 mg/L, 2689-folds of initial engineered yeast. These results indicated that co-optimization of heterologous pathway and chassis metabolism is a good strategy for enforcing microbial overproduction of natural products.

Tomato (Solanum lycopersicum) SlIPT4, encoding an isopentenyltransferase, is involved in leaf senescence and lycopene biosynthesis during fruit ripening

BackgroundLycopene is an important carotenoid pigment in red fruits and vegetables, especially in tomato. Although lycopene biosynthesis and catabolism have been found to be regulated by multiple factors including phytohormones, little is known about their regulatory mechanism. Cytokinins are crucial to various aspects of plant growth. Isopentenyltransferases (IPTs) catalyze the initial rate-limiting step of cytokinins biosynthesis, however, their roles in fruit ripening remain unclear.ResultsHere, the functions of SlIPT4, encoding an isopentenyltransferase, were characterized via RNAi-mediated gene silencing in tomato. As we expected, silencing of SlIPT4 expression resulted in accelerated leaf senescence. However, down-expression of SlIPT4 generated never-red orange fruits, corresponding with a dramatic reduction of lycopene. Among lycopene biosynthesis-related genes, the fact of remarkable decrease of ZISO transcript and upregulation of other genes, revealed that SlIPT4 regulates positively lycopene biosynthesis via directly affecting ZISO expression, and also supported the existence of regulatory loops in lycopene biosynthesis pathway. Meanwhile, the accumulation of abscisic acid (ABA) was reduced and the transcripts PSY1 were increased in SlIPT4-RNAi fruits, supporting the feedback regulation between ABA and lycopene biosynthesis.ConclusionThe study revealed the crucial roles of SlIPT4 in leaf senescence and the regulatory network of lycopene biosynthesis in tomato, providing a new light on the lycopene biosynthesis and fruit ripening.

Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica.

The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y.lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth. Five sites were found to meet these criteria. Expression levels from a GFP expression cassette were consistent when inserted into AXP, XPR2, A08, and D17, with reduced expression from MFE1. The standardized tool is comprised of five pairs of plasmids (one homologous donor plasmid and a CRISPR-Cas9 expression plasmid), with each pair targeting gene integration into one of the characterized sites. To demonstrate the utility of the tool we rapidly engineered a semisynthetic lycopene biosynthesis pathway by integrating four different genes at different loci. The capability to integrate multiple genes without the need for marker recovery and into sites with known expression levels will enable more rapid and reliable pathway engineering in Y.lipolytica.

Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering

BackgroundMicrobial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered.ResultsIn this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts.ConclusionsSaccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0509-4) contains supplementary material, which is available to authorized users.

A spontaneous bud mutant that causes lycopene and β-carotene accumulation in the juice sacs of the parental Guanxi pummelo fruits (Citrus grandis (L.) Osbeck)

Carotenoids are the second-most widespread pigments in nature and play crucial roles in plants, animals and humans. As one of the main fruit crops worldwide, citrus is an important source of carotenoids, and approximately 115 carotenoid compounds occur in citrus fruit. Recently, Red-fleshed pummelo, a spontaneous bud mutant of Guanxi pummelo that accumulate high-level carotenoids in edible portions, was discovered. The fruits, especially the juice sacs of Red-fleshed pummelo are light red in color, which is a feature of great interest to consumers and producers. In this study, the composition, content, characteristics of the lycopene β-cyclase (β-LCY) gene, and expression models of genes involved in the lycopene biosynthetic pathway of Red-fleshed and Guanxi pummelo fruit juice sacs were examined. Lycopene and β-carotene are the main pigments in Red-fleshed pummelo fruits, whereas the contents of these pigments are too low to be detected in Guanxi pummelo fruits. Four of the amino acids deduced from β-LCY cDNA were found to differ between Red-fleshed and Guanxi pummelo. Lycopene and β-carotene accumulation in Red-fleshed pummelo fruits were related to variation in the expression of genes related to the lycopene biosynthetic pathway, which mainly associated with the up-regulated expression of upstream genes and down-regulated expression of downstream genes in the lycopene biosynthesis pathway. This is a first reports of the biochemical and molecular investigation about Red-fleshed fruit mutant in pummelo.

Putative carotenoid genes expressed under the regulation of Shine-Dalgarno regions in Escherichia coli for efficient lycopene production.

Putative genes crtE, crtB, and crtI from Deinococcus wulumiqiensis R12, a novel species, were identified by genome mining and were co-expressed using the optimized Shine-Dalgarno (SD) regions to improve lycopene yield. A lycopene biosynthesis pathway was constructed by co-expressing these three genes in Escherichia coli. After optimizing the upstream SD regions and the culture medium, the recombinant strain EDW11 produced 88mg lycopene g(-1) dry cell wt (780mg lycopene l(-1)) after 40h fermentation without IPTG induction, while the strain EDW without optimized SD regions only produced 49mg lycopene g(-1) dry cell wt (417mg lycopene l(-1)). Based on the optimization of the upstream SD regions and culture medium, the yield of the strain EDW11 reached a high level during microbial lycopene production until now.

Randomized BioBrick Assembly: A Novel DNA Assembly Method for Randomizing and Optimizing Genetic Circuits and Metabolic Pathways

The optimization of genetic circuits and metabolic pathways often involves constructing various iterations of the same construct or using directed evolution to achieve the desired function. Alternatively, a method that randomizes individual parts in the same assembly reaction could be used for optimization by allowing for the ability to screen large numbers of individual clones expressing randomized circuits or pathways for optimal function. Here we describe a new assembly method to randomize genetic circuits and metabolic pathways from modular DNA fragments derived from PCR-amplified BioBricks. As a proof-of-principle for this method, we successfully assembled CMY (Cyan-Magenta-Yellow) three-gene circuits using Gibson Assembly that express CFP, RFP, and YFP with independently randomized promoters, ribosome binding sites, transcriptional terminators, and all parts randomized simultaneously. Sequencing results from 24 CMY circuits with various parts randomized show that 20/24 circuits are distinct and expression varies over a 200-fold range above background levels. We then adapted this method to randomize the same parts with enzyme coding sequences from the lycopene biosynthesis pathway instead of fluorescent proteins, designed to independently express each enzyme in the pathway from a different promoter. Lycopene production is improved using this randomization method by about 30% relative to the highest polycistronic-expressing pathway. These results demonstrate the potential of generating nearly 20,000 unique circuit or pathway combinations when three parts are permutated at each position in a three-gene circuit or pathway, and the methodology can likely be adapted to other circuits and pathways to maximize products of interest.

Ethanol production by repeated batch fermentation of sweet sorghum juice utilizing thermotolerant yeast Kluyveromyces marxianus DBKKUY-103

Lycopene, a tetraterpenoid containing 13 double bonds, has important pharmaceutical and industrial application values. However, unstable supply of raw for extracted from natural host and residue of chemical reagents in chemically synthesized products have limited the quality of lycopene. Engineering microorganism is thus regarded as an alternative and attractive route for lycopene production. To improve the ability of engineered yeast to synthesize lycopene, here we have optimized the lycopene biosynthesis pathway and chassis metabolism. The results have verified that S. cerevisiae CEN.PK2-1C was more suitable for lycopene production because of its strong ability of synthesizing precursors of terpenoids. Compared to use inducible promoter, the use of constitutive promoter controlling pathway expression was more efficient for balancing lycopene synthesis and chassis metabolism. GGPP was identified the rate-controlling metabolite. Through expanding GGPP pool and MVA pathway, lycopene production was thus significantly increased. By citric acid fed-batch fermentation, the yield of lycopene finally reached 115.64 mg/L, 2689-folds of initial engineered yeast. These results indicated that co-optimization of heterologous pathway and chassis metabolism is a good strategy for enforcing microbial overproduction of natural products.

Characterization of lycopene-overproducing E. coli strains in high cell density fermentations

Previous work identified two recombinant strains of Escherichia coli capable of significant lycopene overproduction. These strains were constructed by superimposing the deletion of three genes, selected through combinatorial and systematic searches of the metabolic landscape, onto a previously engineered strain over-expressing critical genes in the lycopene biosynthesis pathway. In this paper, we characterize the performance of these two strains in comparison to the parental, pre-engineered strain. Specifically, high cell density fermentations were performed after identifying optimized putative operating parameters. High oxygen levels and increased pH values were found to be critical for increasing both specific and volumetric product titers. Carbon balances suggest linkages between glutamate, NADPH, formate, and alanine levels with lycopene overproduction. Furthermore, lycopene production reached nearly 220 mg/l from approximately 27 g dry cell weight/l in these reactors, which is the highest value reported to date for E. coli.

Improving lycopene production in Escherichia coli by engineering metabolic control.

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.