The Study Of Cofactor Engineering In Ethanol And 3-hydroxypropionic Acid Metabolic Pathway In Saccharomyces Cerevisiae

Efficient utilization of all available carbon from lignocellulosic feedstock is one of the major challenges preventing economically biofuels production. To enable xylose utilization, our previous studies introduced both cofactor dependent xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and cofactor independent xylose isomerase (XI) pathway respectively in S. cerevisiae and obtained the respiration deficient strains with efficient xylose metabolism capability. However, in the both pathway recombinant strains, the cofactor imbalance caused byproduct glycerol and/or xylitol accumulation and limited the efficient ethanol production. To eliminate the byproduct accumulation, in this study NADH oxidase from Lactococcus lactis was introduced in both XI and XR-XDH pathway recombinant strains, respectively. The expression level of NADH oxidase was optimized by comparing its expression under the control of different promoters and plasmids to decrease the byproduct accumulation without affecting the xylose metabolism. We found that in XI recombinant strains, relative low expression of NADH oxidase under the control of GPD2 promoter in 2μ plasmid decreased glycerol production 84% and increased ethanol yield 8% and the specific growth rate 12%.Differently, in XR-XDH recombinant strains, higher expression of NADH oxidase under the control of TEF1 promoter in centromeric plasmids showed better performance to reduce both xylitoland glycerol production (60% and 83%, respectively) without affecting xylose consumption very significantly.This study demonstratesthat the fine-turned NADH oxidase can decrease the glycerol or/and xylitol production in both XI and XR-XDH recombinant xylose metabolism strains. Different NADH oxidase expression levels were needed for different pathways. Similar cofactor engineering strategy can be employed for other redox dependent metabolites production.3-Hydroxypropionic Acid (3-HP) as a 3-carbon carboxylic acid, have been identified as one of twelve high potential platform chemicalsby the US Department of Energy.It is a very valuable platform chemical intermediates used to produce various compounds such as acrylamide, malonate,1,3-propanediol and acrylic acid. Besides, 3-HP is a starting materials to produce many polymers such as [poly (3-hydroxypropionic acid)] Currently,3-HP isproduced by chemical routes, mainly from oil and other non-renewable resources. Although the technology has been improved, there are stillmany inevitable problems such as high energy consumption, high pollution, byproduct accumulations and separation. On the contrary, the biological production of 3-HP has many advantages such as mild condition and simple operation, therefore have been getting more attention and becoming one of the hot spots today.Biological production of 3-hydroxypropionicacid can be based on glycerol orglucose as substrates. With strong tolerance, mature genetic manipulation and free from phage intrusion, Saccharomyces cerevisiae has become one of the important candidates to produce 3-HP. In addition, S cerevisiaecan also tolerant high concentration of 3-HP, therefore provides a potential platform for 3-HP production.Among various biosynthesis pathways from glucose, malonyl-CoA pathway is a relatively simple pathway.3-HP is produced through malonyl-CoA, which is carboxylated from acetyl-CoA.This pathway doesn’t consume ATP, therefore is a good synthetic pathway. We expressed the malonyl reductase gene from Chloroflexus aurantiacus, and constructed the glucose to 3-HP pathway. The malonyl reductase activity was 0.11 U/mg protein, however, we didn’t detect the product, perhaps due to the low supply of precursor and cofactor imbalance. The pathway cunsume 2 NADPH, produce 2 NADH, and needs cytosolic acetyl-CoA as the precursor to produce malonyl-CoA, then produce 3-HP. To increase the precursor supply, we over-expressed acetyl-CoA carboxylase gene, introduced ATP-citrate lyase, or introduced pyruvate formate lyase in BY5419-A0 strain which is a high pyruvate accumulation strain. In addition, we also introduced NADP+-dependent formate dehydrogenase gene to relieve the cofactor imbalance. The strain construction has been finished, the follow-up experiments are ongoing.