Metabolic engineering of a laboratory-evolvedThermobifida fuscamuC strain for malic acid production on cellulose and minimal treated lignocellulosic biomass

Abstract

Malic acid is mainly used as an acidulant and taste enhancer in the beverage and food industry. Previously, a mutant strain Thermobifida fusca muC, obtained by adaptive evolution was found to accumulate malic acid on cellulose with low yield. In this study, the malic acid synthesis pathway in T. fusca muC was confirmed to be from phosphoenolpyruvate to oxaloacetate, followed by reduction of oxaloacetate to malate. To increase the yield of malic acid by the muC strain significantly, the carbon flux from pyruvate was redirected to oxaloacetate by expressing an exogenous pyruvate carboxylase (PCx) gene from Corynebacterium glutamicum ATCC 13032 in the chromosome of T. fusca muC-16. The yield of malic acid in the engineered strain muC-16 was increased by 47.9% compared to the parent strain muC. The muC-16 strain was then grown on ∼100 g/L cellulose and the highest titer of malic acid was 62.76 g/L by batch fermentation. T. fusca muC-16 strain converted milled corn stover to malic acid with the highest titer of 21.47 g/L with minimal treatment.

Microbial Production of Malic Acid from Biofuel-Related Coproducts and Biomass

“…In S. cerevisiae, it was shown that pyruvate carboxylase and malate dehydrogenase activities were elevated in a strain exhibiting elevated malic acid production [13]. In a strain of the thermophilic soil bacterium, Thermobifida fusca muC, phosphoenolpyruvate is converted to oxaloacetate by phosphoenolpyruvate carboxylase and the oxaloacetate reduced to malate by malate dehydrogenase [5]. Thus far, the microorganisms utilizing the reductive pathway produced the highest malic acid levels and yields [2,[12][13][14].…”

“…Another species of the fungus, namely Penicillium viticola 152 (isolated from a marine environment) was shown to produce 168 g/L calcium malate in a medium containing 140 g/L (w/v) glucose, 40 g/L (w/v) calcium carbonate and 0.5% (v/v) corn steep liquor in a 10 L fermentor (aeration rate of 8 L/min with stirring speed of 300 rpm) for 96 h at 28 • C [4]. Metabolic engineering of various species is also being explored as a way to produce high levels of malic acid under controlled conditions in a fermentor [2,5,12,14,15,24,25]. It was reported that a metabolically engineered A. oryzae ATCC 56747 produced approximately four-fold higher levels of malic acid than did the wild type strain [12].…”

“…Inhibitors within the biooil were found to be responsible for the diminution in malic acid production by the strain [38]. It has been shown that a strain of the filamentous soil bacterium T. fusca muC could produce malic acid from cellulose at 55 • C. The bacterium produces a cellulase allowing it to degrade cellulose over a wide pH range [5]. The presence of yeast extract in the medium stimulated cell growth and malate production at 55 • C at 250 rpm [5].…”

“…In agriculture, malic acid is used to solubilize aluminum phosphate in soil [4]. Annually, the global market ranges from 40,000-60,000 metric tons of malic acid with the growth rate for malic acid increasing by 4% annually [5]. The value of the market has been estimated as $130 million [5].…”

“…Annually, the global market ranges from 40,000-60,000 metric tons of malic acid with the growth rate for malic acid increasing by 4% annually [5]. The value of the market has been estimated as $130 million [5]. It has been reported that the retail price of a pound of malic acid ranges from $1.80 to $2.00 [3].…”