Microbial Production Of Lycopene Research Articles

Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum.

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Metabolic Engineering of Pichia pastoris for High-Level Production of Lycopene.

Lycopene is widely used in cosmetics, food, and nutritional supplements. Microbial production of lycopene has been intensively studied. However, few metabolic engineering studies on Pichia pastoris have been aimed at achieving high-yield lycopene production. In this study, the CRISPR/Cpf1-based gene repression system was developed and the gene editing system was optimized, which were applied to improve lycopene production successfully. In addition, the sterol regulatory element-binding protein SREBP (Sre) was used for the regulation of lipid metabolic pathways to promote lycopene overproduction in P. pastoris for the first time. The final engineered strain produced lycopene at 7.24 g/L and 75.48 mg/g DCW in fed-batch fermentation, representing the highest lycopene yield in P. pastoris reported to date. These findings provide effective strategies for extended metabolic engineering assisted by the CRISPR/Cpf1 system and new insights into metabolic engineering through transcriptional regulation of related metabolic pathways to enhance carotenoid production in P. pastoris.

Metabolic Engineering of Different Microbial Hosts for Lycopene Production.

As a result of the extensive use of lycopene in a variety of fields, especially the dietary supplement and health food industries, the production of lycopene has attracted considerable interest. Lycopene can be obtained through extraction from vegetables and chemical synthesis. Alternatively, the microbial production of lycopene has been extensively researched in recent years. Various types of microbial hosts have been evaluated for their potential to accumulate a high level of lycopene. Metabolic engineering of the hosts and optimization of culture conditions are performed to enhance lycopene production. After years of research, great progress has been made in lycopene production. In this review, strategies used to improve lycopene production in different microbial hosts and the advantages and disadvantages of each microbial host are summarized. In addition, future perspectives of lycopene production in different microbial hosts are discussed.

Biotechnological production of lycopene by microorganisms.

Lycopene is a dark red carotenoid belonging to C40 terpenoids and is widely found in a variety of plants, especially ripe red fruits and vegetables. Lycopene has been shown to reduce the risk of prostate cancer, other cancers, and cardiovascular disease. It is one of the most widely used carotenoids in the healthcare product market. Currently, commercially available lycopene is mainly extracted from tomatoes. However, production of lycopene from plants is costly and environmentally unfriendly. To date, there have been many reports on the biosynthesis of lycopene by microorganisms, providing another route for lycopene production. This review discusses the lycopene biosynthetic pathway and natural and engineered lycopene-accumulating microorganisms, as well as their production of lycopene. The effects of different metabolic engineering strategies on lycopene accumulation are also considered. Furthermore, this work presents perspectives concerning the microbial production of lycopene, especially trends to construct microbial cell factories for lycopene production. KEY POINTS: • Recent achievements in the lycopene biosynthesis in microorganisms. • Review of lycopene biosynthetic metabolism engineering strategy. • Discuss the current challenges and prospects of using microorganisms to produce lycopene.

Systematic Metabolic Engineering of Saccharomyces cerevisiae for Lycopene Overproduction.

Lycopene is widely used in foods, cosmetics, nutritional supplements, and pharmaceuticals. Microbial production of lycopene has been intensively studied. However, there are few systematic engineering studies on Saccharomyces cerevisiae aimed at achieving high-yield lycopene production. In the current study, by employing a systematic optimization strategy, we screened the key lycopene biosynthetic genes, crtE, crtB, and crtI, from diverse organisms. By adjusting the copy number of these three key genes, knocking out endogenous bypass genes, increasing the supply of the precursor acetyl-CoA, balancing NADPH utilization, and regulating the GAL-inducible system, we constructed a high-yield lycopene-producing strain BS106, which can produce 310 mg/L lycopene in shake-flask fermentation, with gene expression controlled by glucose. In optimized two-stage fed-batch fermentation, BS106 produced 3.28 g/L lycopene in a 7 L fermenter, which is the highest concentration achieved in S. cerevisiae to date. It will decrease the consumption of tomatoes for lycopene extraction and increase the market supply of lycopene.

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.

Construction of Saccharomyces cerevisiae cell factories for lycopene production

For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene.