Astaxanthin is a natural carotenoid pigment with tremendous antioxidant activity and great commercial value. Microbial production of astaxanthin via metabolic engineering has become a promising alternative. Although great efforts have been conducted by tuning the heterologous modules and precursor pools, the astaxanthin yields in these non-carotenogenic microorganisms were still unsatisfactory for commercialization, indicating that in addition to targeted tailoring limited targets guided by rationally metabolic design, combining more globe disturbances in astaxanthin biosynthesis system and uncovering new molecular mechanisms seem to be much more crucial for further development. Since combined metabolic engineering with mutagenesis by screening is a powerful tool to achieve more global variations and even uncover more molecular targets, this study would apply a comprehensive approach integrating heterologous module engineering and mutagenesis by atmospheric and room temperature plasma (ARTP) to promote astaxanthin production in Saccharomyces cerevisiae. Here, compared to the strain with β-carotene hydroxylase (CrtZ) from Alcaligenes sp. strain PC-1, involving new CrtZ from Agrobacterium aurantiacum enhanced astaxanthin yield to 1.78-fold and increased astaxanthin ratio to 88.7% (from 66.6%). Astaxanthin yield was further increased by 0.83-fold (to 10.1 mg/g DCW) via ARTP mutagenesis, which is the highest reported yield at shake-flask level in yeast so far. Three underlying molecular targets (CSS1, YBR012W-B and DAN4) associated with astaxanthin biosynthesis were first uncovered by comparative genomics analysis. To be noted, individual deletion of CSS1 can recover 75.6% improvement on astaxanthin yield achieved by ARTP mutagenesis, indicating CSS1 was a very promising molecular target for further development. Eventually, 217.9 mg/L astaxanthin (astaxanthin ratio was 89.4% and astaxanthin yield was up to 13.8 mg/g DCW) was obtained in 5-L fermenter without any addition of inducers. Through integrating rational engineering of pathway modules and random mutagenesis of hosts efficiently, our report stepwise promoted astaxanthin yield to achieve the highest reported one in yeast so far. This work not only breaks the upper ceiling of astaxanthin production in yeast, but also fulfills the underlying molecular targets pools with regard to isoprenoid microbial overproductions.

Due to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially. By introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell. Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.

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. 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. 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.