Efficient production of l-lactic acid by an engineered Thermoanaerobacterium aotearoense with broad substrate specificity

Abstract

Background: Efficient conversion of lignocellulosic biomass to optically pure lactic acid is a key challenge for the economical production of biodegradable poly-lactic acid. A recently isolated strain, Thermoanaerobacterium aotearoense SCUT27, is promising as an efficient lactic acid production bacterium from biomass due to its broad substrate specificity. Additionally, its strictly anaerobic and thermophilic characteristics suppress contamination from other microoragnisms. Herein, we report the significant improvements of concentration and yield in lactic acid production from various lignocellulosic derived sugars, achieved by the carbon flux redirection through homologous recombination in T. aotearoense SCUT27.

Results: T. aotearoense SCUT27 was engineered to block the acetic acid formation pathway to improve the lactic acid production. The genetic manipulation resulted in 1.8 and 2.1 fold increase of the lactic acid yield using 10 g/L of glucose or 10 g/L of xylose as substrate, respectively. The maximum l-lactic acid yield of 0.93 g/g glucose with an optical purity of 99.3% was obtained by the engineered strain, designated as LA1002, from 50 g/L of substrate, which is very close to the theoretical value (1.0 g/g of glucose). In particular, LA1002 produced lactic acid at an unprecedented concentration up to 3.20 g/L using 10 g/L xylan as the single substrate without any pretreatment after 48 h fermentation. The non-sterilized fermentative production of l-lactic acid was also carried out, achieving values of 44.89 g/L and 0.89 g/g mixed sugar for lactic acid concentration and yield, respectively.

Conclusions: Blocking acetic acid formation pathway in T. aotearoense SCUT27 increased l-lactic acid production and yield dramatically. To our best knowledge, this is the best performance of fermentation on lactic acid production using xylan as the sole carbon source, considering the final concentration, yield and fermentation time. In addition, it should be mentioned that the performance of non-sterilized simultaneous fermentation from glucose and xylose was very close to that of normal sterilized cultivation. All these results used the mutant strain, LA1002, indicated that it is a new promising candidate for the effective production of optically pure l-lactic acid from lignocellulosic biomass.

Figure 1

Schematic diagram of the knockout strategy for the pta and ack genes. The pta-ack locus on the T. aotearoense SCUT 27 chromosome, the pBluescript II SK(+) derived knock out plasmid pPuKAd used to disrupt the pta-ack gene locus and the predicted pta-ack gene locus after double cross over integration are shown. The endonucleolytic cleavage sites used in the pPuKAd construction are indicated. The location of the probe and the expected sizes of the fragments detected by southern blot analysis of the genomic DNA digested with Pst I are shown.

Figure 2

Screening and confirmation of pta and ack genes knockout. (A) polymerase chain reaction (PCR) screening using genomic DNA as template. Lane 1, 1 kb DNA ladder (TaKaRa), Lane 2, SCUT27, Lane 3&4, positive isolates. (B) Southern blot analysis of genomic DNA from wild type SCUT27 (Lane 1) and the pta and ack deletion clones, LA1002 (Lane 2) digested with Pst I. The probe with the expected sizes of 486 bp (position shown in Figure 1), hybridized to one 1.2 kb fragment of wild type DNA, and to one 2.2 kb fragment of the mutant DNA.

Figure 3

Fermentation profiles of the SCUT27 and LA1002 strain in 125 mL serum bottles (modified MTC medium containing 10 g/L glucose or xylose as the unique carbon source). Fermentations were performed in triplicate. All the data were derived from three independent experiments. (A) Acetic acid; (B) H₂; (C) Lactic acid; (D) pH; (E) Ethanol; (F) DCW; (G) Residual sugar.

Figure 4

Time profiles of metabolitics using different sugars as the sole carbon source by LA1002. The bacterium was cultivated in serum bottles for 24 hours at 55°C with the initial pH of 6.0. Because the fermentation broth was too turbid to determine OD₆₀₀ before xylan degraded, the value of DCW of LA1002 using beechwood xylan as substrate was not measured. And the residual sugar using dextran T110 and xylan as the carbon source were also not recorded. The data were calculated from two independent experiments. (A) DCW, (B) Lactic acid concentration, (C) Residual sugar, (D) Ethanol concentration, (E) Lactic acid productivity.

Figure 5

pH effects on metabolic parameters of lactic acid production by T. aotearoense LA1002. The values are average of three independent experiments and the error bars represent standard deviation. (A) DCW; (B) Consumed sugar; (C) Lactic acid; (D) Ethanol; (E) Lactic acid yield; (F) Ethanol yield.