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.