Transcriptional fine tuning to improve GDP-L-fucose production
The regulation of the dosage of specific genes is a common strategy in metabolic engineering to modulate the number of enzyme copies in the host strain cell. A commonly employed approach is the combination of plasmids with different gene copy numbers to modulate the metabolic flux of different pathway modules in E. coli and other microorganisms [22–24]. The metabolic pathway leading to GDP-L-fucose is a native pathway in E. coli, but the productivity is extremely low. As an efficient method to regulate the metabolic pathway, combinatorial optimization of gene expression levels based on gene copy number was applied.
Given that GDP-D-mannose is a pivotal precursor in the target pathway, ManB and ManC were placed as the upstream module responsible for converting mannose-1-P into GDP-D-mannose. Gmd and WcaG were placed on the other module for the downstream pathway. More specifically, three plasmids pACYCDuet-1, pCDFDuet-1, and pETDuet-1 with different gene copy numbers (the gene copy numbers were defined as 10, 20, and 40, respectively) were utilized to reconstruct the de novo pathway for GDP-L-fucose production. Thus, via expressing two modules either on high, moderate, or low copy number plasmids, nine recombinant host strains (EWL01-09) were constructed to discover the optimal combination of these two modules (Figure. 2). The product was verified by ESI-MS (Figure. S1).
Shake-flask fermentation results showed that individual overexpression of Gmd and WcaG under different copy number plasmids, resulted in the engineered strains EWL01, EWL04, and EWL07, and remarkably improved the titer of GDP-L-fucose to 4.13, 3.4, and 2.6 mg/L, respectively, after 20 hours of fermentation (Figure. 2). These results indicated that higher expression levels of the downstream module favored more the accumulation of intracellular GDP-L-fucose.
In order to bolster the supplementation of precursor GDP-D-mannose, the upstream pathway was considered, generating the recombinant strains EWL02, EWL03, EWL05, EWL06, EWL08, and EWL09. Interestingly, overexpression of the upstream module under low gene dosage (pACYC-manC-manB) led to higher GDP-L-fucose production compared with that under moderate or high gene dosage (pCDF-manC-manB or pET-manC-manB). For example, the strains EWL03, EWL06, and EWL09 had 162%, 65%, and 58% higher GDP-L-fucose production compared with the strains EWL02, EWL05, and EWL08, respectively. More specifically, the strain EWL03 (harboring plasmids pET-gmd-wcaG and pACYC-manC-manB) gave the highest GDP-L-fucose titer, which reached 5.33 mg/L.
However, the strain EWL08 (harboring plasmids pACYC-gmd-wcaG and pET-manC-manB) accumulated the lowest GDP-L-fucose concentration (0.6 mg/L), indicating that higher expression levels of the upstream module severely inhibited the conversion of mannose-6-P to GDP-L-fucose. Indeed, the catalytic efficiency of ManC was 10-fold higher than that of Gmd, consequently resulting in the accumulation of intermediate metabolites (GDP-D-mannose) and a toxic byproduct (pyrophosphate, PPi), which may adversely influence cell growth and product accumulation.13
Collectively, these results indicated that relatively higher gene dosage of the downstream pathway and lower gene dosage of the upstream pathway resulted in more GDP-L-fucose yield, potential because this combination alleviated the accumulation of intermediate products and facilitated the efficient transformation of GDP-D-mannose into GDP-L-fucose.
Tuning translation initiation rate through RBS engineering to enhance GDP-L-fucose production
Ribosome binding sites (RBSs) engineering is one of the most widely employed strategies for combinatorial fine-tuning of pathways based on diversification of gene translation levels. To further determine the optimal combination of expression levels in the de novo pathway for GDP-L-fucose biosynthesis, RBS engineering was used as a feasible procedure, which has been widely implemented in many other studies [24–27]. Thus, we adjusted the translation initiation rate of both upstream and downstream modules to further balance the supplementation and consumption of GDP-D-mannose in the target metabolic system.
Subsequently, the standard RBS (RBS-32) , the wild type RBS (RBS-WT) of target genes, and the original RBS (RBS-ori) on Duet-1 plasmids were chosen to fine-tune gene expression at the translational level for further improvement of the productivity of GDP-L-fucose (Figure. 3A). In order to define the expression strength of the engineered RBS variants, SDS-PAGE analysis was performed, which rationally characterized the translational activities of the involved RBSs (Figure. S2). The translation strength of the wild type RBSs was distinctly more powerful than that of RBS-ori, while RBS-ori showed higher RBS activity than RBS-32. Each of the module genes was installed under the control of a relatively strong (RBS-WT, represented as 1), moderate (RBS-ori, represented as 2), and weak (RBS-32, represented as 3) RBS. These RBS sequences were paired to generate nine engineered strains to identify the relatively optimal combination (Figure. 3B).
When the Gmd-WcaG module was expressed under the control of relatively weak RBS (RBS-32), the titers of GDP-L-fucose typically decreased, as observed in the strains EWL13, EWL23, and EWL33, which resulted in 2.13, 1.17, and 2.1 mg/L intracellular GDP-L-fucose concentration, respectively (Figure. 3C). These three strains gained a comparatively lower GDP-L-fucose production, probably owing to the accumulation of intermediate metabolites caused by deficient expression of the downstream module.
The strains EWL32 (harboring plasmids pACYC-[RBS-32]-C-[RBS-32]-B and pET-gmd-wcaG) and EWL31 (harboring plasmids pACYC-[RBS-32]-C-[RBS-32]-B and pET-[RBS-WT]-G-[RBS-WT]-W) produced 5.67 mg/L and 6.77 mg/L GDP-L-fucose at 20 h, which corresponds to a 6.4% and 27% increase, respectively, compare to strain EWL03 (5.33 mg/L). In addition, intracellular GDP-L-fucose accumulated to 3.87, 5.16, and 4.9 mg/L in EWL12, EWL21, and EWL11, respectively (Figure. 3C). All these titers were slightly reduced compared with the strain EWL31, indicating that relatively excessive protein expression levels of the upstream module blocked the carbon flux to the desired product.
By expressing the upstream pathway on low RBS activity (RBS-32) and expressing the downstream pathway on high RBS activity (RBS-WT), the levels of GDP-L-fucose produced through the de novo synthesis pathway were more elegantly modulated and achieved an optimal GDP-L-fucose production of 6.77 mg/L in the EWL31 strain.
Effects of inactivating wcaJ on GDP-L-fucose production
Apparently, a common pathway engineering approach to enhance the yield of valuable products is to overexpress the rate-limiting enzymes and inactivate competing pathways to orient the metabolic flux to the pathway of interest. GDP-L-fucose is one of the crucial intermediates for the synthesis of colanic acid . In order to augment the intracellular concentration of GDP-L-fucose, the metabolic flux from GDP-L-fucose to colanic acid was blocked via inactivating the gene wcaJ encoding the UDP-glucose lipid carrier transferase . Specifically, the CRISPR/Cas9 system was implemented to rapidly and efficiently knock out wcaJ in E. coli BL21 (DE3) [30–31].
The shake-flask fermentation showed that the intracellular GDP-L-fucose concentration reached 12.26 mg/L (2.75 mg/g DCW) at 20 h in EWL34 and gained a 1.53-fold improvement compared with that in EWL31. Furthermore, dry cell weight for strain EWL34 was 4.45, illustrating 7% less growth compared to EWL31 (4.79 at 20 h). Therefore, the deletion of wcaJ effectively bolstered GDP-L-fucose titers, and the cell biomass and growth conditions of the engineered strain were not apparently affected (Figure. 4). This result indicated that knocking out wcaJ blocked the flux from GDP-L-fucose to colonic acid and significantly improved the accumulation of the target product.
Effect of facilitating the cofactor NADPH regeneration pathway and GTP biosynthesis pathway on GDP-L-fucose production
Cofactors are involved in large numbers of intracellular reactions and critically manipulate carbon metabolism and redox balance. As the most essential redox carriers corresponding to cellular metabolism, NAD(P)+, NAD(P)H and nucleotides play pivotal roles in the efficient production of biochemicals and pharmaceuticals [32–33]. Coupling cofactor engineering with metabolic flux engineering can increase the efficiency and productivity of large-scale processes for products of interest. For this reason, we regenerated the cofactor systems by strengthening the endogenous cofactor pathway related to the desired metabolite.
Previous studies illustrated that overexpression of zwf gene for NADPH regeneration  or gsk gene for GTP regeneration  was able to optimally enhance the intracellular GDP-L-fucose accumulation than up-regulating other genes in pentose phosphate pathway or the guanosine nucleotides biosynthetic pathway. To further boost the concentration of intracellular GDP-L-fucose, both glucose-6-phosphate dehydrogenase (Zwf) and guanosine-inosine kinase (Gsk) were overexpressed to improve the balance of consumption and regeneration of related cofactors. Figure. 5 illustrates that introduction of cofactor engineering module significantly improved the production of intracellular GDP-L-fucose.
The recombinant E. coli overexpressing Zwf (EWL35) showed 15 mg/L (3.6 mg/g DCW) intracellular GDP-L-fucose accumulation, and thus the titer in this strain was increased by 22.4% compared with that in the EWL34 strain. Furthermore, an even better performance was achieved in the Gsk-overexpressing strain EWL36 (with an increase of 31.7% compared with EWL34), indicating that strengthening the GTP biosynthesis pathway by overexpression of Gsk, which catalyzes the bioconversion of inosine and guanosine to IMP and GMP, respectively , efficiently enhanced GDP-L-fucose production. It is worth noting that the cell growth of strain EWL36 was noticeably boosted by overexpression of Gsk. This might be attributed to the reinforcement of biosynthesis of guanosine nucleotide, an indispensable precursor for the synthesis of nutrient elements such as vitamins and amino acids [25, 35].
Given that ManC demands GTP for GDP-D-mannose biotransformation and that WcaG needs NADPH for the biosynthesis of target products, efficient feeding of these two cofactors is critical for improving production. Thus, we simultaneously overexpressed Zwf and Gsk in strains harboring plasmids for GDP-L-fucose production, generating the strain EWL37. As expected, EWL37 was also amenable to efficiently produce GDP-L-fucose and the accumulation of intracellular GDP-L-fucose reached to 18.33 mg/L (4.5 mg/g DCW), corresponding to a 49.5% increase compared to that in EWL34. These results suggested that the proper cofactor engineering made the production of GDP-L-fucose more efficient.
To obtain high titers of GDP-L-fucose under certain conditions for the engineered strain, fed-batch cultivation of the optimal GDP-L-fucose accumulating strain EWL37 was employed. The fermentation results illustrated that cellular growth was continuously increasing till the end of the fermentation. When the initial glucose was exhausted, glucose feeding started and was limited at a constant flow rate of 8 g glucose per hour to control the production of acetic acid, which could be toxic to cell growth. After the addition of IPTG (the final concentration was 0.1 mM) at 16 h, the product started to accumulate slowly within the first 8 hours. Then, GDP-L-fucose rapidly accumulated with sustained consumption of glucose and oxygen. After 24 h from induction, the production of GDP-L-fucose could be accumulated to 106 mg/L (4.28 mg/g DCW) (Figure. 6). These results indicated that the constant supplementation of carbon source, efficient NADPH and GTP regeneration and metabolic flux toward the desired biosynthesis pathway noticeably boosted the biosynthesis of GDP-L-fucose. Although the titer is not optimal compared with others, the specific GDP-L-fucose content is demonstrably superior to previous 3.5 mg/g DCW, which proved that more efficient cell factories are established in our research.