Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production

Abstract

The central carbon metabolite acetyl-CoA and the cofactor NADPH are important for the synthesis of a wide array of biobased products. Here, we constructed a platform yeast strain for improved provision of acetyl-CoA and NADPH, and used the production of 3-hydroxypropionic acid (3-HP) as a case study. We first demonstrated that the integration of phosphoketolase and phosphotransacetylase improved 3-HP production by 41.9% and decreased glycerol production by 48.1% compared with that of the control strain. Then, to direct more carbon flux toward the pentose phosphate pathway, we reduced the expression of phosphoglucose isomerase by replacing its native promoter with a weaker promoter, and increased the expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by replacing their native promoters with stronger promoters. This further improved 3-HP production by 26.4%. Furthermore, to increase the NADPH supply we overexpressed cytosolic aldehyde dehydrogenase, and improved 3-HP production by another 10.5%. Together with optimizing enzyme expression of acetyl-CoA carboxylase and malonyl-CoA reductase, the final strain is able to produce 3-HP with a final titer of 864.5 mg/L, which is a more than 24-fold improvement compared with that of the starting strain. Our strategy combines the PK pathway with the oxidative pentose phosphate pathway for the efficient provision of acetyl-CoA and NADPH, which provides both a higher theoretical yield and overall yield than the reported yeast-based 3-HP production strategies via the malonyl-CoA reductase-dependent pathway and sheds light on the construction of efficient platform cell factories for other products.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

“…Streamlining sugar utilization through the use of this carbon-conserving pathway has the potential of increasing maximum theoretical yields for AcCoA-derived products in biotechnological applications [23][24][25]. Such a strategy has been harnessed both in bacteria and yeast, most often as an additional route to the linear EMP glycolysis [2,26,27]. The question is whether a synthetic PKT metabolism can be implanted in P. putida to boost AcCoA levels-but such a goal entails a radical rewiring of the metabolic connections that lead to AcCoA formation in this obligate aerobe [28] that runs a cyclic, ED-based sugar catabolism [29,30].…”

O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum

“…Acetyl-CoA is a direct precursor of OAH biosynthesis, and a very key intermediate metabolite and regulator in organisms [ 38 ]. Therefore, the effective supply of acetyl-CoA should be an important factor for the efficient production of OAH.…”

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

“…The PPP pathway and TCA cycle are known to be the major sources of NADPH regeneration in organisms, and glucose 6-phosphate dehydrogenase (ZWF) and 6-phosphogluconate dehydrogenase (GND) are key enzymes involved in NADPH synthesis. Studies have shown that overexpression of zwf and/or gnd can significantly increase the availability of intracellular NADPH [ 16 , 42 ]. In this study, the native promoter of zwf in the recombinant strain BS008 was replaced with the constitutive strong promoter P hbs , resulting in the attainment of the strain BS010.…”