1. INTRODUCTION
Terpenoids are a large family of secondary metabolites that has diverse
biological functions, and thus have a broad market prospect in food,
pharmaceutical and cosmetic industries . Among them, lycopene serves as
an excellent antioxidant applied in food, pharmaceutical and cosmetic
industries [2]. Isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP) are two precursors for terpenoids
synthesis, the flux of which is closely related with the production .
Nowadays, two natural terpenoids synthesis pathways,
2-C-methyl-D-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways,
are commonly utilized .
IUP is a two-step artificial pathway directly supplying two C5 building
blocks through added isoprenol/prenol. It is more competitive because it
costs 2 adenosine triphosphates (ATP) compared with MEP and MVA pathway
respectively having 7 and 6 steps and consuming 3ATP, 3 nicotinamide
adenine dinucleotide phosphate (NADPH) and 3ATP, 2NADPH, which means
it’s more energy efficient. Besides, utilizing isoprenol/prenol as
substrates instead of other carbon source like glucose or glycerol, IUP
is characterized as partly orthogonal in aspect of metabolic substrates
. Recently, IUP has exhibited great potential in synthesis of terpenoids
in different hosts. For example, the introduction of IUP toSaccharomyces cerevisiae elevated the IPP/DMAPP pool by 147-fold
compared with the native pathway . As to Yarrowia lipolytica , IUP
contributed to more than 15.7-fold IPP/DMAPP that of using MVA only . InE.coli , the expression of IUP was capable of converting 2 g/L
prenol to 1.5 g/L geranoids and 0.5 g/L limonene . It was also employed
in E.coli , leading to 248 mg/L β-carotene and 364 mg/L
R-(-)-linalool . Therefore, it is worth applying to greater extent.
It is worth noting that the expression form of IUP is an important
aspect for optimization, mainly including plasmid expression or
integration of the genome. Plasmid expression, however, brings about
many problems. First, the expression level is not stable due to
imprecise copy number controlled by several factors . Second, it adds
extra burden to cells, leading longer lagged phase which further harms
the productivity . Besides, the serious ‘loss of plasmids’ condition
occurred in many cases. For example, during the fed-batch fermentation
of astaxanthin, cells lost plasmids in the exponential phase and it
results in unpromising production . Also, it adds up the fermentation
cost, which isn’t realistic in industrial production . In comparison,
genome multi-position integration is relatively more advantageous . The
lycopene synthetic stability of two strains, respectively employing
plasmid system and genome integration, was compared. The accumulation of
the former one decreased to only 3.3% after 21stgeneration without CmR while the latter one kept the
same level, which highlighted the edge of genome expression compared
with plasmid system . In recent years, editing strategies that employ
CRISPR-associated transposases (CASTs) enabled genome programming and
accelerated the process of construction of engineered cell factories
which was once time-consuming and labor-intensive . With the help of
these tools, strain library with various copies can be rapidly
constructed and screened for the most suitable copy number for desired
purpose. For instance, Zhang et al. developed multicopy chromosomal
integration using the CASTs in E. coli , with which the glucose
dehydrogenase expression cassettes were integrated into the BL21(DE3),
increasing 2.6-fold enzymes than that of strain using pET24a .
Meanwhile, the GDH activity of plasmid-carrying strain began falling
from 24 h while the strain obtained by transposes demonstrated
continuous and stable synthesis during 43 h fermentation period, finally
reaching 2.3-fold that of plasmid-carrying strain . Furthermore,
PtrCASR, an updated version, is relatively more effective, being able to
integrate 15.4kb cargo with 100% integration into multi positions of
the genome, with 12.5% of the total 16 strains tested obtaining 8
copies of cargo after a round of transposition .
In this study, lycopene was chosen as a model terpenoid compound. First,
the feasibility of IUP pathway was verified. Then, the IUP expression
cassette was optimized for effectively accumulation of lycopene, through
promoter engineering, ribosome-binding site (RBS) screening and IPP
Delta-isomerase (IDI) selection. Furthermore, the induction
concentration of isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), the ratio
and concentration of prenol/isoprenol was optimized, respectively.
Besides, the IUP expression cassettes were rapidly integrated into the
genome by the PtrCAST and strain with highest titer was selected
followed by stability test.