On January 10, 2023, Beijing time, Lian Jiachang’s research group of Zhejiang University published a research paper “Biosynthesis of Catharanthine in Engineered Pichia pastoris” in the journal Nature Synthesis, reporting for the first time the use of Pichia cell factory to efficiently synthesize vinblastine.
Following Professor Keasling’s team reporting the breakthrough results of de novo synthesis of vinblastine precursors (vinblastine and vindolin) in Nature, Lian Jiachang’s research group of Zhejiang University published a research paper in Nature Synthesis, reporting for the first time the efficient synthesis of vinblastine using Pichia cell factories. Vinblastine and vindolin can be combined to form the most effective anticancer drug in clinical application – vinblastine. However, the low natural content and complex structure of these plant-derived secondary metabolites hinder their large-scale plant extraction or chemical synthesis, resulting in low yields and high costs. In this study, Pichia yeast was used as the chassis cell to reconstruct the vinblastine synthesis pathway, and the de novo synthesis from a simple carbon source to a high value-added natural product was realized (Figure 1).
Figure 1: De novo synthesis pathway of vinblastine in Pichia yeast
▌1. Screening and characterization of stable integration sites
Firstly, 31 integration sites were screened on the genome of Pichia yeast, and the integration efficiency of each integration site was tested with red fluorescent protein gene as the reporter, and the expression intensity test, stability analysis and growth analysis of the integration site with high integration efficiency were tested. The integration efficiency of 24 of the 31 integration sites reached more than 80%, and there was a correlation between fluorescence intensity and site (Figure 2). After 30 passages of continuous transfer in non-selective medium, all colonies showed red fluorescence, indicating the stability of the heterologous gene expression box in the Pichia genome. In addition, the efficiency of multiplex genome integration was evaluated, and the efficiency of simultaneous integration of three-point points was higher than 60%.
Figure 2: Excavation and characterization of integration sites
▌2. Biosynthesis of isocarboside to vinblastine
Considering the complexity of the vinblastine synthesis pathway, it was divided into three functional blocks (CAN module, STR module and NPT module) for reconstruction and optimization. First, the CAN module (CAN4A) was reconstructed, and the yield of vinblastine was 125 μg/L with exogenous addition of isocarboside (Figures 3A and 3B). CrPAS is a key protein in the CAN module, and its expression by fusion with MBP can effectively increase the yield of vinblastine (MBP3-tPAS) to 270 μg/L (Figure 3C). Due to the lack of detection methods for most of the intermediates in the pathway, by increasing the copy number of the CAN module gene to identify the rate-limiting enzyme of the pathway, it can be concluded that SGD, GS, PAS, DPAS and CS are the main rate-limiting enzymes of the pathway. Increasing the copy number of these rate-limiting enzyme genes on the genome yields strain CAN7, and when 4.5 mg/L isocarboside is added in vitro, vinblastine yields can reach 1.1 mg/L (Figure 3D).
Figure 3: Construction and optimization of CAN modules
▌3. Biosynthesis of vitexol to vinblastine
On the basis of strain CAN7, the STR module is introduced to obtain strain CAN9 (Figure 4A). With the exogenous addition of 150 mg/L of vitexol, the yield of vinblastine was 1.2 mg/L. By increasing the copy number of the STR module gene, CrSTR and CrSLS were found to be the main rate-limiting enzymes, increasing their copy number (CAN11), and the yield of vinblastine was 2.7 mg/L (Figure 4B).
Figure 4: Construction and optimization of STR modules
▌4. De novo biosynthesis of vinblastine
On the basis of strain CAN11, the NPT module was introduced to obtain strain CAN14 (Figure 5A), and the yield of vinblastine was only 1.6 μg/L. The introduction of MLPL (latex protein gene) into strain CAN14, additional introduction of CrG8H and CrGES-ScERG20WW driven by constitutive promoters, overexpression of transcription activator MXR1, the formation of strain CAN17, the yield of vinblastine increased to 20.3 μg/L (Figure 5B). When methanol is the only carbon source and energy source, more than 70% of the carbon metabolic flux flows to the isomorphic pathway (energy and carbon dioxide generation) rather than the assimilation pathway (biosynthesis), so attempts were made to introduce a non-inhibitory carbon source, and it was found that vinblastine yield can be increased to 65 μg/L when methanol is co-cultured with trehalose or mannitol (Figure 5C).
Figure 5: Construction and optimization of the NPT module
▌5. Metabolic engineering of chassis cells
Finally, the chassis cells are modified. Overexpression of Saccharomyces cerevisiae-derived adenosylmethionine synthase increases SAM supply and increases vinblastine yield by ~2-fold (CAN18A). Overexpression of CYB5 increases the electron transport efficiency of P450 and increases the yield of vinblastine by ~1.7 times (CAN18B). Combining these metabolic engineering strategies to construct CAN19 resulted in a ~6-fold increase in vinblastine yield (Figure 6A) and a yield of up to 2.57 mg/L in a 1 L fermenter with fed-batch fermentation (Figure 6B).
Figure 6: Metabolic engineering optimization and high-density fermentation of strains
Taking isocarboside and vitexol as nodes, the vinblastine synthesis pathway was divided into three functional modules, which were integrated and optimized sequentially in Pichia spp. The constructed engineered strain of Pichia CAN19 yielded 0.38 mg/L and 2.57 mg/L on shaker flasks and fermenters, respectively. The vinblastine pathway is the most complex biosynthetic pathway constructed in non-model strains to date, demonstrating the advantages and potential of Pichia as a cell factory for synthetic plant natural products.
This research was supported by the National Key Research and Development Program of China (2018YFA0901800 and 2021YFC2103200), the National Natural Science Foundation of China (22278361), and the Zhejiang Outstanding Youth Fund (LR20B060003). (Source: Science Network)
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