Enhancement of PPP flux by increasing the expression of zwf in different degrees
First, to further optimize the flux ratio of EP-bifido pathway, we aimed to enhance the expression of the first key gene of the PPP, zwf, by replacing its promoter with relative stronger promoters. We selected five constitutive promoters with different strengths from the Anderson promoter library. The theoretical strength of each promoter is shown in Table 3. We compared the actual expression strength of these five synthetic promoters with the original zwf promoter by placing a gfp gene downstream of the promoters and cultivated the engineered strains for fluorescence intensity detection. The fluorescence/OD600 of detected at 16 h is shown in Figure 1A. The strength of the promoters was relatively consistent with that stated by the Anderson promoter library. The strength of promoters BBa-J23100 and BBa-J23104 was relatively strong, and BBa-J23100 was the strongest. The strength of the original (native) zwf promoter is between that of BBa-J23108 and BBa-J23114, and is relatively weak.
To detect the effect of PPP enhancement on MVA production, plasmids pBSA (expressing three enzymes catalysis acetyl-CoA to mevalonate) and pFF (carrying fbp and fxpk gene) were transformed into the five zwf-enhanced strains and cultivated with the control strain BW-P/pBSA pFF (abbreviated to BW-P BF). Strain BW-P10 BF showed almost the same growth and glucose consumption as the others, while the conversion rate of MVA was far higher than that in the control strain due to less byproducts generation. Promoters BBa-J23100 and BBa-J23108 resulted in the highest yield of MVA, 64.3% (mol/mol) and 62.3% (mol/mol) respectively, although the strength of the promoters did not show a perfectly positive correlation with the MVA yield. This proved that enhancing expression of gene zwf was effective for increasing the PPP flux.
13C-Metabolic flux analysis of changes in central carbon metabolism flux and energy metabolism
Strains BW-P10 BF and BW-P08 BF and control strain BW-P BF were chosen for metabolic flux analysis. With the zwf promoter replaced, the normalized data showed that the carbon flux through the oxidative part of the PPP was significantly increased, and the carbon flux through the TCA cycle was decreased, which was consistent with our expectations. More carbon flux moved towards the EP-bifido pathway. The two zwf-expression-enhanced strains showed a large difference in TCA cycle flux, which may explain the growth difference between these strains (Figure 1C).
In addition, the ATP, NADPH, and NADH synthesis capacity and glucose consumption of the three strains were compared based on the 13C-MFA data. After the EP-bifido pathway and the MVA synthetic pathway were introduced, the NADPH content and yield of the strain were significantly improved. pfkA deficiency shunted carbon flux to the PPP and the expression level of zwf was increased. Comparison of the zwf-expression-enhanced strains showed that overexpression of zwf enhanced NADPH synthesis, and the NADPH level was positively correlated with the promoter strength. Taking strains BW25113, BW-P BF, and BW-P10 BF as examples, the introduction of the EP-bifido pathway and overexpression of zwf changed the main source of NADPH: The main NADPH generating pathway shifted from isocitrate dehydrogenase in the TCA cycle to glucose-6-phosphate dehydrogenase in the PPP. This further proved that we have redirected part of the carbon flux of the EMP pathway to the PPP.
In addition, the production of NADH also changed significantly, as shown in Figures 3C and Figures 3D. Through zwf enhancement, the total amount and the yield of NADH decreased significantly. The NADH was produced distinctly in strain BW-P10 BF compared with wild-type strain BW25113: In strain BW25113, five dehydrogenases were the main source of NADH [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase (PDH), malate dehydrogenase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase]; in strain BW-P10 BF, NADH was mainly formed via GAPDH and PDH.
The EP-bifido pathway and MVA synthetic pathway expression increased the ATP yield from glucose, but the total amount of ATP decreased (Figure 3E). The pfkA deficiency greatly impaired the EMP pathway and thereby blocked the three steps of substrate level phosphorylation absorption (glycerate-1, 3-diphosphate, phosphoenolpyruvate kinase, and succinyl CoA synthetase). This can also be verified from the origin ratio of ATP (Figure 3F). Enhancing zwf expression resulted in increased ATP production and yield in strain BW-P08 BF compared with BW-P BF.
Down regulation of EMP pathway flux by targeting pfkA using CRISPRi system
To further rationally use the carbon source, we tried to suppress pfkA in a time-controlled way through exogenous induction and inhibition using CRISPRi system (Figure 4). The CRISPRi gene regulation system requires only two components, dCas9 protein and a gRNA, to achieve regulation of the transcription level of any gene in the genome. The degree of suppression of gene expression can be controlled by adjusting the binding position and expression amount of the gRNA. Thus CRISPRi is widely used in the field of metabolic engineering and had shown a relatively good inhibition effect [26, 27].
To avoid the growth inhibition that may be caused by dCas9 from the CRISPRi gene regulation system, we selected a relatively low strength promoter, BBa-J23134, to promote dcas9. In order to obtain a different repression range, three different sgRNAs targeting the promoter or coding region of pfkA were designed. sgRNA1 were designed on the promoter region of pfkA, sgRNA2 and sgRNA3 targeted the coding chain of pfkA, at the region of 100bp and 200bp downstream of the initial codon, which may cause different repression effect [28]. After dcas9 and the sgRNAs were incorporated into pFF and pBSA respectively, six CRISPRi-regulated strains were generated. The fermentation results showed that the introduction of CRISPRi significantly inhibited the growth of cells and the glucose consumption was also reduced compared with that of the control strain BW25113 zwf-23100 pFF pBSA (abbreviated to BBF). This may be caused by the toxicity or leaky expression of dCas9. The CRISPRi-regulated strains were induced at 12 h by adding IPTG. The fermentation results showed that the three inhibitory sites we selected had different inhibitory effects (Figure 5). sgRNA1 showed a better inhibition effect. Although strain BW25113 pFF-dCas9 pBSA-sgRNA1 produced only 8.53 g/L MVA, its MVA conversion rate reached 68.7%, which exceeded the previous best MVA conversion rate. Four control strains were also constructed to confirm the effect of the CRISPRi system on cell growth. Strains BW25113 zwf-23100 pBSA-sgRNA1 pFF, BW25113 zwf-23100 pBSA pFF-dCas9, BW25113 zwf-23100 pBSA-sgRNA1-dCas9 pFF and BW25113 zwf-23100 pBSA pFF, were abbreviated to BB1F, BBFd, BB1dF, and BBF, respectively. The results proved that CRISPRi can restrain cell growth effectively in these engineered strains (Figure S1). The inhibitory effect of sgRNA1 on_pfkA is suitable for enhancing MVA fermentation in the EP-bifido system (Figure 5). In the CRISPRi-regulated strains, we hardly detected any acetic acid, ethanol, or other byproducts during the fermentation process, which was in line with our expectations. The timely inhibition of pfkA reduces the flux of glycolysis, so there was no excessive carbon source overflow.