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Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Elevation of lipid productivity by enhancing the pentose phosphate pathway in Aspergillus oryzaeKoichi TamanoNational Institute of Advanced Industrial Science and Technology

Research aims

Results

Recently, the focus has shifted to biofuels generatedfrom plant biomass owing to the increasing global demandfor fossil fuels and carbon neutral characteristics of plantbiomass-derived biofuels leading to a lesser effect onglobal environment. The filamentous fungus Aspergillusoryzae can convert biomacromolecules from plant biomassto many varieties of small molecules by both catabolismand assimilation. Thus, A. oryzae has been used to develophigh production systems for lipids, such as free fatty acids(FFAs) and acylglycerol, as source materials of biofuelsand chemical products.1), 2) In this study, genes encodingenzymes of the pentose phosphate pathway (PPP), whichgenerates cytosolic NADPH required for lipid biosynthesis,were subjected to overexpression. Consequently, an increase in the FFA productivity was expected.

The FFA productivity of A. oryzae has been increased bymodifying its metabolism by metabolic engineering techniques. Previously, the acyl-CoA synthetase gene faaA disruptant (ΔfaaA) had been identified to most increase itsFFA productivity (9.2-fold increase compared to the parental RIB40 strain).2) To further increase FFA productivity,the PPP was selected as a modification target because itgenerates cytosolic NADPH required for lipid biosynthesis.
Because whole transcriptome data of A. oryzae metabolismimply that insufficient synthesis of cytosolic NADPH limitsFFA production, enhancement of the PPP was consideredeffective in further increasing FFA productivity. Therefore,individual genes encoding enzymes composing the PPPwere overproduced for this enhancement.
Genes encoding enzymes functioning in the PPP wereselected based on a previous study on a genome scale metabolic model of A. oryzae3) and on homology search resultsfor identified PPP enzyme-encoding genes of Saccharomyces cerevisiae by BLASTP. A list of selected genes encoding enzymes composing the PPP is described in Fig. 1.
Overexpression mutants of the selected genes were constructed from ΔfaaA_pyrG- using pyrG as a selectionmarker. FFA productivities of the constructed mutants weremeasured after liquid culture. Consequently, overexpression of AO090023000345, which was predicted to encodetransketolase, caused a 1.4-fold increase in FFA productivity (Fig. 2A). Total dry cell weight of hyphae increased by1.2 fold in the AO090023000345-overexpressing faaA disruptant (ΔfaaA_tktOE) than in ΔfaaA. Therefore, by multiplying these increase rates, the total FFA production yieldper unit volume of culture increased by 1.7 fold due tooverexpression (Fig. 2B).
Interestingly, in the liquid culture, ΔfaaA_tktOE did notform hyphal clusters. The ΔfaaA hyphae formed clusters,while the ΔfaaA_tktOE hyphae appeared to be dispersed inthe culture medium (Fig. 3).
On the other hand, overexpression of the genes encodingother enzymes in the PPP did not cause an increase in FFAproductivity. In addition, except for AO090023000345, twogenes were predicted to encode transketolase in A. oryzae.
However, overexpression of these genes also did not increase the FFA productivity.
Together with the PPP, the malic enzyme reaction, whichMethodsOverexpression mutants for genes encoding enzymescomposing the PPP were constructed from the A. oryzaefaaA disruptant with a defective pyrG (ΔfaaA_pyrG-). Native promoters were replaced with the tef1 promoter(Ptef1), which has constitutively high transcriptional activity. For promoter replacement, DNA fragments harboring,in order, a 1-kb fragment of the 5′-UTR of the replacedgene, the pyrG marker cassette, a 1028-bp Ptef1 fragment,and a 1-kb fragment from a start codon of the replaced genewere prepared by fusion PCR using KOD-PLUS DNApolymerase (Toyobo). Transformants were subjected to single-spore isolation, followed by clone check using PCR.
The constructed overexpression mutants were cultured in50 mL Czapek-Dox liquid medium (modified to contain10% glucose) at 30°C at 200 rpm for 120 hours. The cultured hyphae were used to quantify intracellular FFAs. Hyphae were washed twice by 100 mL Milli-Q water and lyophilized overnight. Using approximately 50 mg lyophilized hyphae, FFAs were extracted in chloroform after disruption with a beads cell disrupter MS-100 (TOMY). Extracted FFAs were precipitated by evaporation. The precipitate was dissolved in 6% Triton X-100/6% ethanol, and5 μL solution was used for FFA quantification by the FreeFatty Acids, Half Micro Test kit (Roche).

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Fig. 1. Illustration of cytosolic NADPH generating metabolism (left), and a list of genes of A. oryzae that encode enzymes predicted to function in thepentose phosphate pathway (right).

mutants for genes (AO090011000876, AO090038000621)predicted to encode malic enzyme were constructed fromΔfaaA_pyrG-. However, the FFA productivity did not increase in these mutants.
Conclusion

Fig. 2. FFA productivity (A) and the production yield (B) of ΔfaaA_tktOE relative to those of ΔfaaA.

By overexpression of the predicted transketolase geneAO090023000345 of the PPP in the A. oryzae faaA disruptant, FFA productivity was increased by 1.4 fold. In addition, the hyphal biomass increased by 1.2 fold. Consequently, overexpression of this gene resulted in a 1.7-foldincrease in FFA production yield per liter of liquid culture.
However, overexpression of genes encoding enzymes directly involved in NADPH synthesis, such as glucose6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, did not contribute to an increasein FFA productivity.
References

Fig. 3. A photograph of the liquid cultured ΔfaaA (left) and ΔfaaA_tktOE (right) in petri dish.

results in pyruvate synthesis from malate derived from theTCA cycle, generates cytosolic NADPH. Overexpression

1) Tamano, K. et al. (2013) Increased production of fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fattyacid synthesis-related genes. Appl. Microbiol. Biotechnol. 97: 269–281.
2) Tamano, K. et al. (2015) Increased production of free fatty acids inAspergillus oryzae by disruption of a predicted acyl-CoA synthetasegene. Appl. Microbiol. Biotechnol. 99: 3103–3113.
3) Vongsangnak, W., Olsen, P., Hansen, K., Krogsgaard, S., andNielsen, J. (2008) Improved annotation through genome-scale metabolic modeling of Aspergillus oryzae. BMC Genomics 23: 245.