Abstract
3-Hydroxypropionic acid (3-HP) has been recognized as one of the top value-added building block chemicals, due to its numerous potential applications. Over the past decade, biosynthesis of 3-HP via the malonyl-CoA pathway has been increasingly favored because it is balanced in terms of ATP and reducing equivalents, does not require the addition of costly coenzymes, and can utilize renewable lignocellulosic biomass. In this study, gene mcr encoding malonyl-CoA reductase from Chloroflexus aurantiacus was introduced into Corynebacterium glutamicum ATCC13032 to construct the strain Cgz1, which accumulated 0.30 g/L 3-HP. Gene ldhA encoding lactate dehydrogenase was subsequently deleted to eliminate lactate accumulation, but this decreased 3-HP production and greatly increased acetate accumulation. Then, different acetate utilization genes were overexpressed to reuse the acetate, and the best candidate Cgz5 expressing endogenous gene pta could effectively reduce the acetate accumulation and produced 0.68 g/L 3-HP. To enhance the supply of the precursor acetyl-CoA, acetate was used as an ancillary carbon source to improve the 3-HP production, and 1.33 g/L 3-HP could be produced from a mixture of glucose and acetate, with a 2.06-fold higher yield than from glucose alone. Finally, to inhibit the major 3-HP competing pathway-fatty acid synthesis, 10 μM cerulenin was added and strain Cgz5 produced 3.77 g/L 3-HP from 15.47 g/L glucose and 4.68 g/L acetate with a yield of 187 mg/g substrate in 48 h, which was 12.57-fold higher than that of Cgz1. To our best knowledge, this is the first report on engineering C. glutamicum to produce 3-HP via the malonyl-CoA pathway. The results indicate that the innocuous biosafety level I microorganism C. glutamicum is a potential industrial 3-HP producer.
Highlights
IntroductionAs an important building block chemical with wide applications in the chemical, food, agricultural and pharmaceutical industries [1,2,3], 3-hydroxypropionic acid (3-HP) has been designated as one of Catalysts 2020, 10, 203; doi:10.3390/catal10020203 www.mdpi.com/journal/catalysts
As an important building block chemical with wide applications in the chemical, food, agricultural and pharmaceutical industries [1,2,3], 3-hydroxypropionic acid (3-HP) has been designated as one of Catalysts 2020, 10, 203; doi:10.3390/catal10020203 www.mdpi.com/journal/catalystsCatalysts 2020, 10, 203 the top value-added chemicals by the U.S Department of Energy (DOE) both in 2004 and 2010 [1,2].To avoid the serious environmental problems of unsustainable 3-HP synthesis from petrochemicals, increasing attention has been paid to microbial production of 3-HP from renewable feedstocks, such as glycerol, glucose, xylose or even fatty acids [4,5,6]
The reason might be that lactate was redirected to pyruvate, while acetate was converted to acetyl-CoA [33,37], which is a direct precursor of malonyl-CoA, and more favorable for 3-HP production
Summary
As an important building block chemical with wide applications in the chemical, food, agricultural and pharmaceutical industries [1,2,3], 3-hydroxypropionic acid (3-HP) has been designated as one of Catalysts 2020, 10, 203; doi:10.3390/catal10020203 www.mdpi.com/journal/catalysts. The imbalance of reducing power would bring a heavy burden to cellular metabolism [9] To avoid these shortcomings, the malonyl-CoA pathway and β-alanine pathway were proposed as alternative routes, since both are balanced in terms of ATP and reducing equivalents, independent of costly coenzyme additives, and can utilize renewable lignocellulosic biomass [9,10,11]. Corynebacterium glutamicum is one of the most important amino acid industrial producers with the GRAS (generally regarded as safe) status It has been widely used for the production of various chemicals, fuels and materials over the last decades [23,24], and exhibited excellent performance in the bioconversion of organic acids [25,26]. 3-hydroxypropionic acid; ldhA, lactate dehydrogenase A; ackA, acetate kinase A; pta, acetyl phosphate transferase;acc, acc,acetyl-CoA acetyl-CoAcarboxylase; carboxylase;mcr, mcr,malonyl-CoA malonyl-CoAreductase; reductase;fasA, fasA,fatty fattyacid acidsynthetase synthetaseA.A
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