Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory.

Parthenolide, a kind of sesquiterpene lactone, is the direct precursor for the promising anti-glioblastoma drug ACT001. Compared with traditional parthenolide source from plant extraction, de novo biosynthesis of parthenolide in microorganisms has the potential to make a sustainable supply. Herein, an integrated strategy was designed with P450 source screening, nicotinamide adenine dinucleotide phosphate (NADPH) supply, and endoplasmic reticulum (ER) size rewiring to manipulate three P450s regarded as the bottleneck for parthenolide production. Germacrene A oxidase from Cichorium intybus, costunolide synthase from Lactuca sativa, and parthenolide synthase from Tanacetum parthenium have the best efficiency, resulting in a parthenolide titer of 2.19 mg/L, which was first achieved in yeast. The parthenolide titer was further increased by 300% with NADPH supplementation and ER expanding stepwise. Finally, the highest titers of 31.0 mg/L parthenolide and 648.5 mg/L costunolide in microbes were achieved in 2.0 L fed-batch fermentation. This study not only provides an alternative microbial platform for producing sesquiterpene lactones in a sustainable way but also highlights a general strategy for manipulating multiple plant-derived P450s in microbes.

Endogenous 2µ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, conventional 2µ-plasmid based plasmid (pC2µ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2µ plasmid (pE2µ) brings the issues concerning plasmid stability and copy number of pC2µ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2µ and built the pE2µ multi-copy system by insertion of the target DNA element and elimination of the original pE2µ plasmid. The resulting plasmid pE2µRAF1 and pE2µREP2 demonstrated higher copy number and slower loss rate than a pC2µ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2µRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2µ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2µ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2µRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2µ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2µ due to the more stable pE2µ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

Metabolic Engineering of Saccharomyces cerevisiae for Enhanced Dihydroartemisinic Acid Production.

Direct bioproduction of DHAA (dihydroartemisinic acid) rather than AA (artemisinic acid), as suggested by previous work would decrease the cost of semi-biosynthesis artemisinin by eliminating the step of initial hydrogenation of AA. The major challenge in microbial production of DHAA is how to efficiently manipulate consecutive key enzymes ADH1 (artemisinic alcohol dehydrogenase), DBR2 [artemisinic aldehyde Δ11(13) reductase] and ALDH1 (aldehyde dehydrogenase) to redirect metabolic flux and elevate the ratio of DHAA to AA (artemisinic acid). Herein, DHAA biosynthesis was achieved in Saccharomyces cerevisiae by introducing a series of heterologous enzymes: ADS (amorpha-4,11-diene synthase), CYP71AV1 (amorphadiene oxidase), ADH1, DBR2 and ALDH1, obtaining initial DHAA/AA ratio at 2.53. The flux toward DHAA was enhanced by pairing fusion proteins DBR2-ADH1 and DBR2-ALDH1, leading to 1.75-fold increase in DHAA/AA ratio (to 6.97). Moreover, to promote the substrate preference of ALDH1 to dihydroartemisinic aldehyde (the intermediate for DHAA synthesis) over artemisinic aldehyde (the intermediate for AA synthesis), two rational engineering strategies, including downsizing the active pocket and enhancing the stability of enzyme/cofactor complex, were proposed to engineer ALDH1. It was found that the mutant H194R, which showed better stability of the enzyme/NAD+ complex, obtained the highest DHAA to AA ratio at 3.73 among all the mutations. Then the mutant H194R was incorporated into above rebuilt fusion proteins, resulting in the highest ratio of DHAA to AA (10.05). Subsequently, the highest DHAA reported titer of 1.70 g/L (DHAA/AA ratio of 9.84) was achieved through 5 L bioreactor fermentation. The study highlights the synergy of metabolic engineering and protein engineering in metabolic flux redirection to get the most efficient product to the chemical process, and simplified downstream conversion process.

Metabolic engineering of Saccharomyces cerevisiae for 7-dehydrocholesterol overproduction.

BACKGROUND: 7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin D3. Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae, the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ24-dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation. RESULTS: In order to decouple 7-DHC production with cell growth, inducible GAL promoters was employed to control 7-DHC synthesis. Meanwhile, the precursor pool was increased via overexpressing all the mevalonate (MVA) pathway genes (ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20). Through screening DHCR24s from eleven tested sources, it was found that DHCR24 from Gallus gallus (Gg_DHCR24) achieved the highest 7-DHC production. Then 7-DHC accumulation was increased by 27.5% through stepwise fine-tuning the transcription level of Gg_DHCR24 in terms of altering its induction strategy, integration position, and the used promoter. By blocking the competitive path (ΔERG6) and supplementing another copy of Gg_DHCR24 in locus ERG6, 7-DHC accumulation was further enhanced by 1.07-fold. Afterward, 7-DHC production was improved by 48.3% (to 250.8 mg/L) by means of deleting NEM1 that was involved in lipids metabolism. Eventually, 7-DHC production reached to 1.07 g/L in 5-L bioreactor, which is the highest reported microbial titer as yet known. CONCLUSIONS: Combined engineering of the pathway and the host cell was adopted in this study to boost 7-DHC output in the yeast. 7-DHC titer was stepwise improved by 26.9-fold compared with the starting strain. This work not only opens large opportunities to realize downstream de novo synthesis of other steroids, but also highlights the importance of the combinatorial engineering of heterologous pathway and host to obtain microbial overproduction of many other natural products.

Design and chemical synthesis of eukaryotic chromosomes.

Following the discovery of the DNA double helix structure and the advancement of genome sequencing, we have entered a promising stage with regard to genome writing. Recently, a milestone breakthrough was achieved in the chemical synthesis of designer yeast chromosomes. Here, we review the systematic approaches to the de novo synthesis of designer eukaryotic chromosomes, with an emphasis on technologies and methodologies that enable design, building, testing and debugging. The achievement of chemically synthesized genomes with customized genetic features offers an opportunity to rebuild genome organization, remold biological functions and promote life evolution, which will be of great benefit for application in medicine and industrial manufacturing.

Chassis and key enzymes engineering for monoterpenes production.

Microbial production of monoterpenes is often limited by their cytotoxicity and in vivo conversion. Therefore, alleviating cytotoxicity and reducing conversion by chassis engineering are highly desirable. On the other hand, engineering key enzymes is also critical for improving monoterpenes production through facilitating the biosynthesis process. Here we critically review recent advances in cytotoxicity alleviation, reducing in vivo conversion, selecting geranyl diphosphate synthase and engineering monoterpene synthases. These achievements would lead to the development of superior chassis with improved tolerance to cytotoxicity and rationally tailored metabolites profiles to improve titer, yield and productivity for the production of monoterpenes by microbial cells.

"Perfect" designer chromosome V and behavior of a ring derivative.

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.

Engineered biosynthesis of natural products in heterologous hosts.

Natural products produced by microorganisms and plants are a major resource of antibacterial and anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene, pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products.