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.