Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step

Abstract

The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.

Figure 1

Metabolic pathway for PLA-like polymer production in engineered C. glutamicum . PLA-like polymers contained <1 mol% 3HB units. L-LDH, L-lactate dehydrogenase; D-LDH, D-lactate dehydrogenase; PCT, propionyl-CoA transferase; PhaC1STQK, lactate-polymerizing PHA synthase. The bold letters indicate exogenous enzymes. The dashed lines indicate the putative pathways. The dotted line indicates a very weak pathway. The gray ovals indicate significantly pooled metabolites.

Figure 2

Concentrations of the CoA derivatives in C. glutamicum during PLA-like polymer production. The cells were harvested at 18 h. The data is the average of six independent samples along with the standard deviation. Gray: acetyl-CoA, white: lactyl-CoA.

Figure 3

SDS-PAGE analysis of C. glutamicum . Whole cell extracts were applied. The arrow indicates the size of PhaC1STQK. 1: Purified His-tag fusion of PhaC1STQK (0.072 μg), 2: size marker, 3: wild type (5.4 μg), 4: recombinant form harboring the parent PHA synthase gene phaC1STQK (5.0 μg), 5 : recombinant form harboring the codon-optimized ephaC1STQK gene (4.0 μg).