4. DISCUSSION
The present work showed a successful extension of the GA biosynthesis step by expressing a newly identified functional CYP5139G1 from G. lucidum in S. cerevisiae . Such work is certainly important to unveil the mystery of the GA post-modifications in G. lucidum . It is also critical to synthesize GAs in yeast chassis by synthetic biology. Our work of mining enzymes involved in GA biosynthesis and pathway reconstruction in S. cerevisiae has proved the feasibility of integrating gene mining with synthetic biology to produce GAs from the simple sugar glucose. Interesting, although there have been over 150 GAs reported from G. lucidum (Baby et al., 2015), the DHLDOA biosynthesized here was a new one. This may be because DHLDOA was quickly converted into the downstream GAs, failing to accumulate inG. lucidum . In another aspect, in the yeast cells, DHLDOA was accumulated due to the lack of subsequent enzymes that can catalyze DHLDOA. The work demonstrated that synthetic biology approach could not only produce known GAs, but also synthesized unnatural new GAs.
Generally, with the extension of biosynthetic steps, the end metabolite accumulation will dramatically decrease, which is a great challenge to natural product production by synthetic biology approach (Cravens et al., 2019). To increase the product titer is critical to determine its chemical structure with sufficient amount and high purity as well as to characterize the function of related enzyme(s). Up to now, many metabolic engineering strategies have been applied to improve the yield of target products, such as copy number engineering, promoter engineering, pathway enzyme engineering, co-factor engineering, and so on (Lian et al., 2018). Regulation of gene expression levels by adjusting plasmid copy number is a convenient and efficient approach to achieve a high accumulation of target natural products (Lan et al., 2019; Lian et al., 2016). Similar to a recent study (Lan et al., 2019), we used a dual tunable plasmid system to optimize the balanced expression of CYP5150L8 and CYP5139G1.
It can be observed that the copy number of the plasmid pRS426-KanMX-CYP5139G1 at H100G500 was more than that at H100G300, but the transcription level of CYP5139G1 did not increase (Fig. 6c, 6d), suggesting that the expression of the corresponding gene did not continue to increase with the increasing copy number when it exceeded a certain level. This may be due to that not only the copy number but also the transcription efficiency could affect the gene expression ( Liu et al., 2020; Sha et al., 2013; Zhang et al., 2018). In addition, with the addition of the Hyg and G418, the cell growth was impaired (Fig. 6a). This may be due to 1) the toxicity of antibiotics (Balibar et al., 2016), 2) metabolic burden caused by high plasmid copy number and high level of foreign gene expression (Fig. 6c, 6d) on the cells (Hasunuma et al., 2015; Lan et al., 2019; Z. Liu et al., 2013), and/or 3) toxicity of the synthesized product (Qiao et al., 2019; Zhu et al., 2018). The problems of metabolic burden and product toxicity may be also the reason why the DHLDOA production decreased with the increased copy number of pRS426-KanMX-CYP5139G1 (Fig. 6b). It was worth noting that by adopting the dual tunable plasmid system to adjust the ratio of CYP5139G1 to CYP5150L8 expressions, the DHLDOA production reached a maximum value of 2.2 mg/L at H100G300. In another aspect, at H0G300, the production of DHLDOA was not so high (0.5 mg/L), which may be due to insufficient supply of the substrate HLDOA. The results showed that the gene copy number adjustment was effective but had its limitation. Taking strategies including engineering at the mRNA level (Curran et al., 2013; Du et al., 2012), engineering at the protein level (Forman et al., 2018; Ignea et al., 2018), modifying suitable chassis cells or compartmentalization to accommodate the heterologous enzymes and so on (Cravens et al., 2019; Lian et al., 2018) may further improve the catalytic efficiency of CYP5139G1. In addition, considering the effect of CPR on CYP activity (Sugishima et al., 2014), and the mutual influence of multiple CYPs (Bassard et al., 2012; Xiao et al., 2019), pairing novel CPR, individually regulating the relative levels of CPR and CYPs and constructing efficient metabolon (multi-CYPs and CPR complex) (Gou et al., 2018; Laursen et al., 2016; S. Z. Wang et al., 2017), these measures may have the potential to achieve high catalytic efficiency for sequence reactions and to improve DHLDOA production.
In summary, here we succeeded in extending the GA biosynthetic step from HLDOA into producing a new GA, DHLDOA, by expressing CYP5139G1 fromG. lucidum in the HLDOA producing S. cerevisiae . The DHLDOA production titer was significantly enhanced by modulating plasmid copy numbers. The study will be helpful to future extension of GA synthetic pathway, and it also helps to elucidate the authentic GA synthetic pathway in G. lucidum, as recently demonstrated by combining such work (Wang et al., 2018) with CRISPR/Cas9 based knocking-out of the gene CYP5150L8 (Wang et al., 2020). The study also laid a good foundation for future efficient biomanufacturing of various GAs using the established yeast chassis.