Construction of lactic acid overproducing Clostridium thermocellum through enhancement of lactate dehydrogenase expression

Abstract

Rapid expansion of global market of lactic acid (LA) has prompted research towards cheaper and more eco-friendly strategies for its production. Nowadays, LA is produced mainly through fermentation of simple sugars or starchy biomass (e.g. corn) and its price is relatively high. Lignocellulose could be an advantageous alternative feedstock for LA production owing to its high abundance and low cost. However, the most effective natural producers of LA cannot directly ferment lignocellulose. So far, metabolic engineering aimed at developing microorganisms combining efficient LA production and cellulose hydrolysis has been generally based on introducing designer cellulase systems in natural LA producers. In the present study, the approach consisted in improving LA production in the natural cellulolytic bacterium Clostridium thermocellum DSM1313. The expression of the native lactate dehydrogenase was enhanced by functional replacement of its original promoter with stronger ones resulting in a 10-fold increase in specific activity, which resulted in a 2-fold increase of LA yield. It is known that eliminating allosteric regulation can also increase lactic acid production in C. thermocellum, however we were unable to insert strong promoters upstream of the deregulated ldh gene. A strategy combining these regulations and inactivation of parasitic pathways appears essential for developing a homolactic C. thermocellum.

Current Progress in Production of Building-Block Organic Acids by Consolidated Bioprocessing of Lignocellulose

“…Improvement of LA production in natural (hemi)cellulolytic microorganisms is at its infancy. Metabolic engineering studies on a number of (hemi)cellulolytic bacteria such as C. thermocellum [90,91], C. bescii [92], Thermoanaerobacter mathranii [93], T. saccharolyticum [94], and Thermoanaerobacterium thermosaccharolyticum [95] have suggested promising strategies to enhance LA accumulation in these strains. In most cases they consisted in disruption of fermentative pathways that compete with LA biosynthesis for reducing equivalents (e.g., production of H 2 ), carbon substrates (e.g., production of acetate), or both (production of ethanol and formate) (reviewed by [7]).…”

“…In most cases they consisted in disruption of fermentative pathways that compete with LA biosynthesis for reducing equivalents (e.g., production of H 2 ), carbon substrates (e.g., production of acetate), or both (production of ethanol and formate) (reviewed by [7]). Alternative strategies focused on increasing expression of lactate dehydrogenase (Ldh) [91,92] or engineering the redox state of the cell [96][97][98]. So far, the highest LA titer (7.9 g/L) was obtained through cellobiose fermentation by a C. thermocellum strain in which ethanol production had been disrupted (by deletion of its main alcohol/aldehyde dehydrogenase AdhE), and featuring a mutant Ldh, which is independent from allosteric activation by fructose 1,6-bisphosphate (FBP) (Table 2) [90,91,99].…”

“…Alternative strategies focused on increasing expression of lactate dehydrogenase (Ldh) [91,92] or engineering the redox state of the cell [96][97][98]. So far, the highest LA titer (7.9 g/L) was obtained through cellobiose fermentation by a C. thermocellum strain in which ethanol production had been disrupted (by deletion of its main alcohol/aldehyde dehydrogenase AdhE), and featuring a mutant Ldh, which is independent from allosteric activation by fructose 1,6-bisphosphate (FBP) (Table 2) [90,91,99]. About 85% of the maximum theoretical yield was obtained, but at low volumetric productivity (0.2 g/L/h).…”

Thermoanaerobacter Species: The Promising Candidates for Lig-nocellulosic Biofuel Production

“…Among microorganisms that can ferment biomass to biofuels, thermophilic anaerobic bacteria, with an optimal growth temperature range of 45-70 °C, have some advantages over mesophilic bacteria [21], such as the ability to co-utilize glucose and xylose by Thermoanaerobacter species [22,23] (they are further subdivided into the genus Thermoanaerobacter and the genus Thermoanaerobacterium due to some differences such as the absence or presence of the nitrogenase genes [24]), lower risk of the contamination for high temperature fermentation [25], less heat exchange following pretreatment and higher economy to recover ethanol at temperatures over 50 °C by continuous distillation [26]. Another common and well-studied thermophile is Clostridium thermocellum, which could degrade cellulose but the titer of biofuels produced was low [27][28][29]. Here, we mainly focus on Thermoanaerobacter species, which might be the potential chassis microorganisms for lignocellulosic biorefinery with the ability to directly utilize hemicellulose or even cellulose [30,31].…”

Genome-Wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

“…The bacterium’s unique features and its industrial relevance in the conversion of biomass (lignocellulose) to a chemical or fuel draws increasing interest to understand various aspects of this microorganism. In the past decade, research related to this cellulose-degrading bacterium flourished and there are numerous studies on the bacteria’s physiology and metabolism ( Xiong et al, 2016 ; Olson et al, 2017 ; Dash et al, 2019 ; Jacobson et al, 2020 ), cellulosome genesis ( Yoav et al, 2017 ; Kahn et al, 2020 ), genetic tools development ( Olson and Lynd, 2012 ; Marcano-Velazquez et al, 2019 ; Walker et al, 2020 ) genome engineering for improving the conversion of lignocellulose to a target product ( Hon et al, 2017 ; Mazzoli et al, 2020 ; Tafur Rangel et al, 2020 ), computational modeling related to this microbe ( Dash et al, 2017 ; Garcia et al, 2020 ), and beyond.…”