Construction of Synthetic POM Cycles
Gene cassettes encoding four distinct POM cycles were constructed in the Yeast/E.coli shuttle vector YCplac111 (YCpNC1-4; Table 1). Each cassette contains a truncated version of the S. cerevisiae malic enzyme gene MAE1, designated sMAE1, lacking the first 90 nucleotides which encode amino acids believed to effect targeting to the mitochondria . Lacking this signaling sequence it is expected that sMae1 will localize to the cytosol. The cassettes also contain the gene sequences for either of the endogenous S. cerevisiae isoforms of pyruvate carboxylase (PYC1 or PYC2) and an isoform of malate dehydrogenase (‘MDH1 or ‘MDH2) resulting in four combinatorial arrangements (Figure 1B).
Differing from most other eukaryotic cells, S. cerevisiae encodes two isoenzymes of pyruvate carboxylase that are localized to the cytosol rather than a single mitochondrial enzyme. Expression of the isoforms is differentially regulated at the level of transcription depending on factors such as growth phase, carbon source and nitrogen source [25-27], however, in the gene cassettes the coding sequences were placed under the regulation of an HXT7 promoter to eliminate differential transcriptional regulation. However, the protein isoforms display different allosteric properties with respect to acetyl-CoA activation and aspartate inhibition  with Pyc1 displaying a higher degree of cooperativity. In addition, Pyc2 reportedly displays lower abundance and reduced activity in glucose . Therefore, it is reasonable to predict that these isoforms may display distinct differences when incorporated into the synthetic POM cycles.
For the expression of malate dehydrogenase, we aimed to utilize versions that would be both cytosolic and stable in medium containing glucose as the carbon source. To achieve this, a nucleotide sequence corresponding to the 17 amino acid mitochondrial signal sequence from MDH1  was deleted in order to avoid targeting to the mitochondria. Mdh2 is subject to both transcriptional repression and phosphorylation-mediated post-translational degradation in the presence of glucose ; therefore, to stabilize Mdh2, the nucleotide sequence encoding 12 amino acids at the amino-terminus was deleted in the overexpression constructs used in this study. Control plasmids consisting of each of the genes individually expressed were also generated. The proposed mechanism of carbon shuttling and generation of NADPH by these shunts is shown in Figure 1 along with the normal localization and proposed altered localization of the overexpressed forms.
Verification of protein isoform expression
cerevisiae strains harboring vectors for expression of the individual PYC1, PYC2, MDH1 and MDH2 variants were assayed by western blot to confirm accumulation of the respective protein products. Pyc1 and Pyc2 were both expressed in the test strains but Pyc1 accumulated to a higher level than Pyc2 (Fig 2). The open reading frames of PYC1 and PYC2 were both under the regulation of an HXT7 promoter, which was expected to limit the differences in transcriptional regulation between the two but qPCR analysis demonstrated that the mRNA for PYC2 accumulated to higher levels than the mRNA for PYC1 (Fig. 3B). Thus, these results indicate that either Pyc1 is translated more efficiently or is more stable than Pyc2. This is similar to the distinctly higher abundance and activity of the endogenous Pyc1 when cells are cultured in medium with glucose as carbon source . In the context of utilizing a synthetic POM cycle to generate NADPH, the stoichiometry of the enzyme activities is likely to be as important as the total activity for optimal function. In this case, high pyruvate carboxylase activity may not be optimal for operation of the POM cycle unless it can be matched by the activity of the malate dehydrogenase and malic enzyme. The Mdh1 and Mdh2 variants we tested displayed similar abundance to one another supporting the contention that the amino-terminal truncation of Mdh2 would stabilize the enzyme when cells were fermenting glucose as a carbon source. It is also worth noting that both malate dehydrogenase variants display steady state abundance similar to Pyc1 (Fig 2). Interestingly, when mRNA was monitored in strains expressing the full POM cycle vectors, we observed that strains overexpressing ‘MDH1 saw a concurrent increase in mRNA from the endogenous MDH2 while this did not appear to be true for cells overexpressing ‘MDH2 (Fig. 3A). There is no published report of Mdh1 influencing the expression of MDH2 but it is unlikely that this would be detrimental to the functioning of the synthetic POM cycle. The sMAE1 allele was constant between the four POM cycles and levels of the expression of this enzyme were not evaluated.
Effect of Synthetic POM cycles on Fatty Alcohol Production
As increasing NADPH production is extremely salient for the production of oleochemicals in microbial systems, we investigated the effects of the four synthetic POM cycles in strains minimally engineered towards fatty alcohol production. These strains (BMY12 - BMY16; Table 1) overexpress FAS1 under regulation of the strong constitutive PGK1 promoter (PPGK1), express a codon-optimized version of the Mus musculus fatty acyl-CoA reductase (ScFAR) and express a synthetic POM cycle. Increased expression of FAS1 results in a coinciding increase in the levels of FAS2 yielding an overall increase in the expression of the FAS complex . This increases fatty acid biosynthesis activity, ultimately creating an increased demand for NADPH in the cells. As the reactions catalyzed by both the FAS complex and ScFAR1 enzyme are NADPH-dependent (Figure 1) we hypothesized the availability of NADPH may become limiting in this synthetic pathway and that expression of the synthetic POM cycles would increase availability of NADPH and drive increased flux through the pathway resulting in an increased titer of fatty alcohols.
Seven independent colonies from each transformation (BMY12 – BMY17) were tested for fatty alcohol production in shake flask culture. There was a high degree of variability in the titer of fatty alcohols produced among individual transformants carrying the same plasmid, particularly in those strains that produced the highest levels of fatty alcohols (Figure S3). Upon further investigation, we found that the variation among transformants harbouring the same plasmid was largely accounted for by some colonies being high producers while others were low producers (Fig S3). The production characteristic of a transformant was clonal. When a high producing colony was streaked to isolate independent colonies all of those plasmid-bearing colonies consistently maintain high levels of production. Three single colonies were isolated from each of the highest producing candidates from the strains (BMY12 - BMY17) and tested for fatty alcohol production (Figure 3A). The BMY13 strain (PYC1, ‘MDH2, sMAE1) displayed an increase in fatty alcohol production relative to the other synthetic POM cycles and the controls. Specifically, BMY13 increased fatty alcohol production by 40% from the control strain BMY12. resulting in production titers of 68.6 ± 3.3 mg/L and 49.0 ± 2.2 mg/L respectively. Interestingly, an ~ 20% decrease in fatty alcohol production was observed for the PYC2 overexpressing strains BMY15 and BMY16, producing 36.8 ± 1.2 mg/L and 38.3 ± 3.7 mg/L respectively, however this decrease in productivity can be accounted for by the lower cell density. When normalized for culture density BMY15 (PYC2, ‘MDH2, sMAE1) and BMY16 (PYC2, ‘MDH1, sMAE1) produced fatty alcohol similar to the vector control strain (Figure S4). Overexpression of sMAE1, or either isoform of PYC or MDH alone (BMY17 - BMY21) showed no increase in fatty alcohol production relative to the empty vector control strain (BMY12; Figure S5).
Although the YCpNC1 plasmid (PYC1, ‘MDH2, sMAE1) in strain BMY13 was the only synthetic POM cycle to result in increased fatty alcohol production in our FAS overexpressing background, all combinations of the POM cycle enzymes should be capable of increasing NADPH availability to support elevated fatty alcohol production and so we opted to test the effects of the synthetic POM cycles in a second strain background. We went on to investigate whether the synthetic POM cycles would be capable of increasing fatty alcohol production in a zwf1D strain expressing ScFAR (BMY22 - BMY26). As the zwf1D strain cannot generate NADPH through the pentose phosphate pathway (Figure 1) - one of the primary sources of NADPH production in actively proliferating S. cerevisiae - it is expected that fatty alcohol production in these strains may be improved by alternate sources of NADPH production, such as the synthetic POM cycles. Again, we observed that strains producing higher titers of fatty alcohols had greater variability in production between individual colony replicates (Figure 4, BMY22-BMY24 vs BMY25 and BMY26). Strains containing the plasmids YCpNC1 (PYC1, ‘MDH2, sMAE1) and YCpNC2 (PYC1, ‘MDH1, sMAE1) (BMY23 and BMY24, respectively; Table 1) displayed increased fatty alcohol production when compared to the strains containing YCpNC3 (PYC2, ‘MDH2, sMAE1) and YCpNC4 (PYC1, ‘MDH1, sMAE1), BMY25 and BMY26 produced lower levels of fatty alcohols (Figure 4). Production of fatty alcohols by BMY24, however, was comparable to the control strain BMY22. This trend remained true when the effects of biomass accumulation were also taken into account and fatty alcohol production was expressed as mg/L/OD (Figure S6).
Although all combinations of the POM cycle enzymes should be capable of increasing NADPH availability resulting in elevated fatty alcohol production, we found that only YCpNC1 (PYC1, ‘MDH2, sMAE1) had appreciable effects on fatty alcohol production in both the FAS overexpressing and zwf1Δ strains. As such, it may be that some combinations of POM cycle enzymes lead to unexpected metabolic consequences or increased metabolic burden with little commensurate benefit to redox cofactor biosynthesis. This appears to be especially true of strains overexpressing PYC2 (BMY15, BMY16, BMY25 and BMY 26; Figures 3 and 4), where both reduced cell growth (Figure 3) and decreased fatty alcohol production were observed (Figures 3 and 4). Due to this, we conclude that strains overexpressing PYC1 as part of a synthetic POM cycle outperformed those overexpressing PYC2. Why Pyc1 out performs Pyc2 in these cycles is not clear. It may simply be that Pyc1 accumulates to higher levels than Pyc2 (Figure 2) or that the relative abundance of Pyc1 and the Mdh expressed results in more stoichiometric expression. However, we cannot discount the possibility that the functional differences between the isoforms in this context is due to allosteric regulation by cellular metabolites or other inherent differences between to two protein isoforms.
The POM cycle employing Pyc1 Mdh2 sMae1 yielded higher fatty alcohol production than did the Pyc1 Mdh1, sMae1 cycle (Figures 3 and 4). This demonstrates that Mdh2 is more effective in the context of this cycle. The coding sequences for the two isoforms were modified to provide stable cytosolic expression in glucose and showed comparable protein abundance in actively proliferating cells (Figure 2); however, Mdh2 is native to the cytosol whereas Mdh1 normally functions within the environment of the mitochondria with a different redox environment. It may also be significant that Mdh2 displays a higher affinity for NADH that does Mdh1, which may make it more effective in driving the oxaloacetate to malate reaction . Another isoform of malate dehydrogenase, the peroxisome associated Mdh3 has also been employed in a POM cycle . However, Mdh3 has a significantly lower affinity for oxaloacetate than does Mdh2 (300µM compared to 70µM for Mdh2) suggesting it might not be the best enzyme to drive a POM cycle for NADPH synthesis.
The early reports on application of synthetic POM cycles for metabolic engineering in S. cerevisiae all employ the endogenous Pyc2 isoform, along with Mdh2 and Mae1. They have been reported to counter the redox imbalance of a heterologous xylose fermentation pathway  and for the production of isobutanol in S. cerevisiae . However, while the expression of the cycle in xylose fermenting conditions was reported to lead to an increased rate of xylose consumption, ultimately it resulted in decreased ethanol production . When implemented in isobutanol producing strains, a version of the cycle expressing a truncated Mae1 (sMae1p) expected to locate to the cytosol, was found to increase isobutanol production in a strain that overexpressed the Ehrlich pathway and ILV2 but was found to have no effect on isobutanol titers in other strains . While the efficacy of these POM cycles is not immediately clear [12, 14-16], it is possible that they may be improved by overexpressing Pyc1 rather than Pyc2 as we have shown that enzyme combinations including Pyc1, particularly when combined with Mdh2, out perform those containing Pyc2. Adding support to this argument, a recent report converting S. cerevisiae to lipogenic metabolism utilized a Pyc1 containing POM cycle with success, however alternate combinations were not reported .
Effect of NAD Kinase expression on fatty alcohol production
An alternative to the use of synthetic POM cycles to increase NADPH, is to express enzymes with NAD kinase activity. To determine whether overexpression of NAD kinase activity could improve fatty alcohol biosynthesis, YEF1, UTR1 and a cytosolic version of POS5, denoted POS5c were introduced into a high-copy plasmid harbouring ScFAR1 and these constructs were installed in S. cerevisiae. Overexpression of UTR1 (BMY27) resulted in a slight increase in fatty alcohol production over the control strain BMY12, increasing from 82 to 90 mg/L (Figure S7) or 7.23 mg/L/OD to 8.13 mg/L/OD (Figure 5). Overexpression of cytosolic POS5c (BMY29) resulted in a significant increase in fatty alcohol titers from 82 mg/L to 111 mg/L or 7.23 mg/L/OD to 11.95 mg/L/OD (Figures S7 and Figure 5). Overexpression of YEF1, on the other hand, failed to improve titers. On their own we found that only Pos5c was truly effective at increasing fatty alcohol production and that the increase in production was comparable to expressing the best POM cycle (PYC1, ‘MDH2, sMAE1). In this independent trial, we found that expressing YCpNC1 in strain BMY13, produced 119 mg/L fatty alcohols while expressing Pos5c in strain BMY29 resulted in 111 mg/L fatty alcohols. Compared to the vector control strain BMY12 (OD600 = 11.5), both BMY29 and BMY13 displayed a slight decrease in biomass accumulation OD600 = 9.3, 10.04 respectively (Figure S7A). The fact that Pos5c was the only NAD kinase to show a significant increase in fatty alcohol production is likely due to the fact that mitochondrial Pos5 is a NADH kinase that phosphorylates NADH preferentially with much lower activity towards NAD+. Yef1 and Utr1 are paralogs that both are located in the cytosol and display activity towards both NAD+ and NADH substrates.
We next sought to investigate whether expression of the NAD kinases and our synthetic POM cycle could be combined to further increase fatty alcohol production. When combined with YCpNC1 encoding POM cycle enzymes PYC1, ‘MDH2, and sMAE1, the overexpression of YEF1 and UTR1 had similar effects as they did when expressed with the empty vector control YCplac111 (Figure 5). UTR1 overexpression combined with YCpNC1 (BMY30) resulted in a slight increase in fatty alcohol production compared to the control expressing YCpNC1 (BMY13) (125 mg/L vs. 119 mg/L or 12.6 mg/L/OD vs. 11.87 mg/L/OD). The YEF1 overexpressing strain BMY31 resulted in a slight decrease in fatty alcohol production compared to BMY13. These strains BMY30, BMY31 also saw the same slight decrease in cell density associated with the YCpNC1 carrying control strain (BMY13). The decrease in growth associated with these strains may be due to the fact that both NAD kinases and the Pyc1 in the POM cycle are ATP-dependent, and therefore effective generation of NADPH to drive fatty alcohol production will occur at the expense of ATP driven processes. Interestingly, combining the overexpression of POS5c and YCpNC1 into strain BMY32 did not result in synergistic or even additive effects on fatty alcohol production. Fatty alcohol production in BMY32 was less than in the cells expressing either Pos5c alone or the Pyc1, ‘Mdh2, sMae1 POM cycle (BMY13). The decrease in fatty alcohol production in this strain is also associated with a reduced cell density suggesting extensive metabolic burden or a negative effect caused by the combination of overexpressed gene products.