Background: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. Results: In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH-or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. Conclusions: Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

BackgroundThe oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest.ResultsIn this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized.ConclusionTaken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0909-8) contains supplementary material, which is available to authorized users.

Limonene, a plant-derived natural cyclic monoterpene, is widely used in the pharmaceutical, food, and cosmetics industries. The conventional limonene biosynthetic (CLB) pathway in engineered Saccharomyces cerevisiae consists of heterologous limonene synthase (LS) using endogenous substrate geranyl diphosphate (GPP) and suffers from poor production of limonene. In this study, we report on an orthogonal engineering strategy in S. cerevisiae for improving the production of limonene. We reconstructed the orthogonal limonene biosynthetic (OLB) pathway composed of SlNDPS1 that catalyzes IPP and DMAPP to NPP (cis-GPP) and plant LS that converts NPP to limonene. We find that the OLB pathway is more efficient for production of limonene than the CLB pathway. When expression of the competing gene ERG20 was chromosomally regulated by the glucose-sensing promoter HXT1, the OLB pathway-harboring strain produced 917.7 mg/L of limonene in fed-batch fermentation, a 6-fold increase of the CLB pathway, representing the highest titer reported to date. Orthogonal engineering exhibits great potential for production of terpenoids in S. cerevisiae.

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Microbial production of steroid drugs exhibits great potentials in much greener and more sustainable manners, in which engineering multiple cytochrome P450s is the prerequisite requirement. The pairing of multicomponents of P450 systems is a tremendous challenge. Herein, biosynthesis of pregnenolone (a common precursor of steroid drugs) in Yarrowia lipolytica was taken as a typical instance to explore the engineering strategy of the cytochrome P450 side-chain cleavage enzyme (P450scc) system. The mature forms of the components belonging to P450scc system, CYP11A1, adrenodoxin (Adx), and adrenodoxin reductase (AdR), were coexpressed in a former constructed campesterol producing strain. To maximize pregnenolone production, an integrative components pairing strategy was proposed for pairing the component sources and balancing the expression levels of CYP11A1, Adx, and AdR. Led by the above approaches, a 2344-fold improvement of pregnenolone titer was achieved at the shake flask level. Consequently, a highest reported pregnenolone titer of 78.0 mg/L in microbes was obtained in a 5 L bioreactor. Our study not only highlights the integrative components pairing of cytochrome P450scc as a general strategy for engineering other cytochrome P450s, but also provides a feasible and efficient platform of Y. lipolytica for other steroids production.