Enhanced isoprenoid production through the cofactor metabolic engineering in

Saccharomyces cerevisiae

Linqi Zhu, Huiqing Chong, Chi Bun Ching
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore
117585

Abstract:

NADPH, a reducing equivalent, usually plays an important role in the biosynthetic reactions and the anabolic pathways in various microorganisms. The isoprenoid pathway in Saccharomyces cerevisiae is largely dependent on the availability of NADPH pools, and therefore increment in NADPH availability is likely to enhance the isoprenoid production.
In this study, we attempt to increase the availability of NADPH to the isoprenoid pathway in S. cerevisiae through integration of NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (gapC) from Clostridium acetobutylicum. To further enhance the NADPH availability, gapC is overexpressed together with (i) native NADH kinase directed to the cytosol (truncated cPOS5) which mediate the ATP- driven conversion from NADH to NADPH, (ii) pyridine nucleotide transhydrogenase (udhA) from Klebsiella pneumoniae for the reversible transfer of reducing equivalents between NADH and NADPH, or (iii) glucose 6-phosphate dehydrogenase (ZWF1) to increase NADPH regeneration from pentose phosphate pathway The effect of increasing NADPH availability on the isoprenoid pathway was investigated through production of amorpha-4,11-diene.
It was found that integration of gapC into the genome of S. cerevisiae alone resulted in a 11.9 % increase in amorpha-4,11-diene yield, while overexpression of gapC and cPOS5 further enhanced the yield to 5.78 mg/l, an 20.9% improvement as compared to the wild-type (4.78 mg/l). However, when gapC was overexpressed with either udhA or ZWF1, the amorpha-4,11-diene yield was found to be even lower than the wild-type.
The host characteristics and product yield changes were closely linked to the variation of the intracellular concentration of the cofactors. These results will provide valuable suggestions for the cofactor regeneration and the design of the metabolic networks to improve the isoprenoid production in yeast.