Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli

Abstract

Background: Lignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40% of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.

Results: The Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara, leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (<2 mg g(-1) h(-1)) compared to carbon excess (25 mg g(-1) h(-1)), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g(-1)) during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g(-1) h(-1)) and to increase the yield of 3HB/cell dry weight to 1.38 g g(-1). Nitrogen-limited fed-batch process design led to further increased specific productivity (38 mg g(-1) h(-1)) but also to additional cell growth (Y3HB/CDW=0.16 g g(-1)). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.

Conclusions: It was demonstrated that by using E. coli PPA652ara, it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB reported here from glucose, xylose and arabinose by E. coli is further comparable to the current state of the art for production from glucose sources.

Figure 1

3HB pathway introduced into E. coli by plasmid pJBG. Genes t3 (acetoacetyl-CoA thiolase) and rx (acetoacetyl-CoA reductase) were cloned from H. boliviensis into plasmid pJBG (pACYC184-derived) to produce pJBGT3RX (shown in figure by the gene order t3-rx) and pJBGRXT3 (gene order rx-t3). The plasmids were subsequently transformed into E. coli AF1000 and PPA652ara and used to produce 3HB in the medium. Conversion of 3HB-CoA into 3HB is likely catalyzed by the E. coli native enzyme thioesterase II (TesB).

Figure 2

Growth of E. coli AF1000 (A) and PPA652ara (B) on glucose, xylose and arabinose in continuous mode without product formation. Parameters: cell dry weight (CDW, filled circles), glucose (Glc, open circles), xylose (Xyl, open squares), arabinose (Ara, open triangles) and acetic acid (HAc, filled squares). Each dilution rate was tested in duplicate and the mean values of the two sample series are represented as dashed lines.

Figure 3

Production of 3HB in E. coli AF1000 (A) and PPA652ara (B) during cultivation on glucose, xylose and arabinose in continuous mode. Parameters: cell dry weight (CDW, filled circles), glucose (Glc, open circles), xylose (Xyl, open squares), arabinose (Ara, open triangles), acetic acid (HAc, filled squares), 3HB (filled triangles) and specific productivity of 3HB (q_3HB, open diamonds). Each dilution rate was tested in duplicate. In A, the dotted and solid lines each represent one sample series. In B, the mean values of the two sample series are represented as dashed lines.

Figure 4

Production of 3HB in E. coli AF1000 (A) and PPA652ara (B) during cultivation on glucose (AF1000), or glucose, xylose and arabinose (PPA652ara) in batch mode. Parameters: cell dry weight (CDW, filled circles), glucose (Glc, open circles), xylose (Xyl, open squares), arabinose (Ara, open triangles), acetic acid (HAc, filled squares), 3HB (filled triangles) and specific productivity of 3HB (q_3HB, open diamonds). The specific productivity of 3HB has been curve fitted to a 1st order polynomial.

Figure 5

Production of 3HB in E. coli AF1000 (A) and PPA652ara (B) during cultivation on glucose, xylose and arabinose in batch mode during nitrogen depletion. Parameters: cell dry weight (CDW, filled circles), glucose (Glc, open circles), xylose (Xyl, open squares), arabinose (Ara, open triangles), acetic acid (HAc, filled squares), 3HB (filled triangles), ammonia (NH₃, filled diamonds) and specific productivity of 3HB (q_3HB, open diamonds). The specific productivity of 3HB in the nitrogen depletion phase has been curve fitted to a 1st order polynomial.

Figure 6

Production of 3HB in E. coli AF1000 (A) and PPA652ara (B) during cultivation on glucose, xylose and arabinose in nitrogen-limited fed-batch mode. Parameters: cell dry weight (CDW, filled circles), glucose (Glc, open circles), xylose (Xyl, open squares), arabinose (Ara, open triangles), acetic acid (HAc, filled squares), 3HB (filled triangles), ammonia (NH₃, filled diamonds) and specific productivity of 3HB (q_3HB, open diamonds). The specific productivity of 3HB has been curve fitted to a 1st order polynomial.