[Heterologous biomimetic synthesis of active ingredients in traditional Chinese medicine:a new mode for protection and development of traditional Chinese medicine resources].

Heterologous biomimetic synthesis of the active ingredients of traditional Chinese medicine(TCM) is a new mode of resource acquisition and has shown great potential in the protection and development of TCM resources. According to synthetic biology and by constructing biomimetic microbial cells and imitating the synthesis of active ingredients in medicinal plants and animals, the key enzymes obtained from medicinal plants and animals are scientifically designed and systematically reconstructed and optimized to realize the heterologous synthesis of the active ingredients in microorganisms. This method ensures an efficient and green acquisition of target products, and also achieves large-scale industrial production, which is conducive to the production of scarce TCM resources. Additiona-lly, the method playes a role in agricultural industrialization, and provides a new option for promoting the green and sustainable deve-lopment of TCM resources. This review systematically summarized the important progress in the heterologous biomimetic synthesis of TCM active ingredients from three research areas: biosynthesis of terpenoids, flavonoids, phenylpropanoids, alkaloids and other active ingredients, key points and difficulties in heterologous biomimetic synthesis, and biomimetic cells with complex TCM ingredients. This study facilitated the application of new generation of biotechnology and theory to the development of TCM.

[Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae].

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Artificial Diploid Escherichia coli by a CRISPR Chromosome-Doubling Technique.

Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR-based chromosome-doubling technique is designed to first construct an artificial diploid Escherichia coli cell. The stable single-cell diploid E. coli is isolated by both maximal dilution plating and flow cytometry, and confirmed with quantitative PCR, fluorescent in situ hybridization, and third-generation genome sequencing. The diploid E. coli has a greatly reduced growth rate and elongated cells at 4-5 µm. It is robust against radiation, and the survival rate after exposure to UV increased 40-fold relative to WT. As a novel life form, the artificial diploid E. coli is an ideal substrate for research fundamental questions in life science concerning polyploidy. And this technique may be applied to other bacteria.

A CRISPR-based chromosomal-separation technique for Escherichia coli.

BACKGROUND: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. RESULTS: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli0.10/4.54 and E. coli2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli0.10/4.54 was quite stable, the two chromosomes of E. coli2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. CONCLUSION: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.

[Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae].

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

[Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae].

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production.

Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.

[Advances in microbial synthesis of plant natural products].

Plant natural products are one of the main sources of small molecule drugs, nutraceuticals, cosmetics and fragrances, and play an important role in economy development. At present, the way of obtaining plant natural products mainly depends on direct extraction from plants, which is farm land occupying and time consuming. The contents of active ingredients in plants are usually low, and thus the production cost is high. By elucidating the biosynthetic pathways and reconstructing the pathways in microbial cells, plant natural products can be produced by fermentation using renewable raw materials. Microbial biosynthesis provides a new route for the supply of plant natural products. This review summarizes the research progress of microbial synthesis of terpenoids, flavonoids, phenylpropanoids and other important natural products of plants in Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Current research challenges and future prospects are also briefly discussed.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants.

BACKGROUND: Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. RESULTS: In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). CONCLUSIONS: All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides.

Eukaryotic yeasts have a variety of subcellular compartments and are ideal platform strains for the construction of complex heterologous natural product biosynthesis pathways. Improving the synthesis efficiency of microbial cell factories through the utilization and modification of subcellular compartments by synthetic biology has good application prospects. Here, we used the yeast PLN1 protein to target the normally endoplasmic reticulum (ER)-localized cytochrome P450 enzyme protopanaxadiol (PPD) synthase (PPDS) to lipid droplets (LDs), which are the storage organelles of the PPDS substrate dammarenediol-II (DD). The efficiency of converting DD to PPD was significantly increased by 394%, and the conversion rate of DD increased from 17.4% to 86.0%. Furthermore, increasing the volume of LDs can significantly enhance the production of DD and its derivatives, but the change in the ratio of the volume and surface area of LDs decreased the conversion efficiency of DD to PPD. Additionally, the biosynthetic pathways of the PPD-type saponin ginsenoside compound K (CK) was reconstituted in a PPD-producing chassis strain, and CK production reached 21.8 mg/L/OD, 4.4-fold higher compared to the native ER-expression strategy. Next, we enhanced the expression of the Pn3-29 gene module to further reduce the accumulation of PPD and increase the production of CK to 41.3 mg/L/OD. Finally, the CK titer of the resulting strain reached 5 g/L in 5 L fed-batch fermentations. This study provides a new strategy for engineering yeast to produce complex natural products.

The origin and evolution of the diosgenin biosynthetic pathway in yam.

Diosgenin, mainly produced by Dioscorea species, is a traditional precursor of most hormonal drugs in the pharmaceutical industry. The mechanisms that underlie the origin and evolution of diosgenin biosynthesis in plants remain unclear. After sequencing the whole genome of Dioscorea zingiberensis, we revealed the evolutionary trajectory of the diosgenin biosynthetic pathway in Dioscorea and demonstrated the de novo biosynthesis of diosgenin in a yeast cell factory. First, we found that P450 gene duplication and neo-functionalization, driven by positive selection, played important roles in the origin of the diosgenin biosynthetic pathway. Subsequently, we found that the enrichment of diosgenin in the yam lineage was regulated by CpG islands, which evolved to regulate gene expression in the diosgenin pathway and balance the carbon flux between the biosynthesis of diosgenin and starch. Finally, by integrating genes from plants, animals, and yeast, we heterologously synthesized diosgenin to 10 mg/l in genetically-engineered yeast. Our study not only reveals the origin and evolutionary mechanisms of the diosgenin biosynthetic pathway in Dioscorea, but also introduces an alternative approach for the production of diosgenin through synthetic biology.

Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli.

This study engineered ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from ß-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L-1 without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of ß-carotene ketolase and ß-carotene hydroxylase were improved for conversion of ß-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.

[Optimization of UDP-glucose supply module and production of ginsenoside F1 in Saccharomyces cerevisiae].

Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.

GuUGT, a glycosyltransferase from Glycyrrhiza uralensis, exhibits glycyrrhetinic acid 3- and 30-O-glycosylation.

Glycyrrhiza uralensis is a well-known herbal medicine that contains triterpenoid saponins as the predominant bioactive components, and these compounds include glycyrrhetinic acid (GA)-glycoside derivatives. Although two genes encoding UDP-glycosyltransferases (UGTs) that glycosylate these derivates have been functionally characterized in G. uralensis, the mechanisms of glycosylation by other UGTs remain unknown. Based on the available transcriptome data, we isolated a UGT with expression in the roots of G. uralensis. This UGT gene possibly encodes a glucosyltransferase that glycosylates GA derivatives at the 3-OH site. Biochemical analyses revealed that the recombinant UGT enzyme could transfer a glucosyl moiety to the free 3-OH or 30-COOH groups of GA. Furthermore, engineered yeast harbouring genes involved in the biosynthetic pathway for GA-glycoside derivates produced GA-3-O-ß-D-glucoside, implying that the enzyme has GA 3-O-glucosyltransferase activity in vivo. Our results could provide a frame for understand the function of the UGT gene family, and also is important for further studies of triterpenoids biosynthesis in G. uralensis.

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11ß-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11ß-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid.

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

Identification of a novel cytochrome P450 enzyme that catalyzes the C-2α hydroxylation of pentacyclic triterpenoids and its application in yeast cell factories.

C-2α hydroxylated triterpenoids are a large class of plant secondary metabolites. These compounds, such as maslinic, corosolic and alphitolic acid, have important biological activities against HIV, cancer and diabetes. However, the biosynthesis pathways of these compounds have not been completely elucidated. Specifically, the cytochrome P450 (CYP) enzyme responsible for C-2α hydroxylation was unknown. In this study, a novel CYP enzyme that catalyzes C-2α hydroxylation was identified in Crataegus pinnatifida (Hawthorn) using a metabolic engineering platform. It is a multifunctional enzyme with C-2α oxidase activity on oleanane-, ursane- and lupane-type pentacyclic triterpenoids. In addition, the complete biosynthesis pathways of these three triterpenoids were reconstituted in yeast, resulting in the production of 384, 141 and 23 mg/L of maslinic, corosolic and alphitolic acid, respectively. This metabolic engineering platform for functional gene identification and strain engineering can serve as the basis for creating alternative pathways for the microbial production of important natural products.