Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol

Abstract

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

Keywords: Kluyveromyces marxianus; Lactobacillus plantarum; Yarrowia lipolytica; ethanol; heterologous protein production; lactic acid; protein expression platform; raw starch; raw starch digesting enzymes.

Figure 1

Time-course of SoAMY-TlGAMY proteins production, utilization of glycerol and synthesis of metabolites in fed-batch production cultures of Y. lipolytica GGY215. Letter codes: GLY, glycerol; CA, citric acid; ERY, erythritol; MAN, mannitol; A-KG, alpha-ketoglutarate; DCW, dry cellular biomass; AU, amylolytic activity in supernatant assayed by microSIT assay. Values indicate means ± SD from four independent runs.

Figure 2

Electrophoretic separation of the protein fractions. Proteins were separated in 15% SDS-PAGE. M: PageRuler™ Prestained Protein Ladder (ThermoScientific, Waltham, MA, USA), 1, 8, 10: supernatant after ammonium sulfate precipitation, 2, 9, 11: resuspended protein after ammonium sulfate precipitation (crude preparation), 3: F-T, 4: W-U, 5: Fraction 1 (F1) with increased Abs280 during affinity chromatography, 6: F2 with increased Abs280 during affinity chromatography, 7: F3 with increased Abs280 during affinity chromatography, 12, 13: culture medium supernatant.

Figure 3

Time course of (A) dp1 release from raw rice starch by SoAMY-TlGAMY crude preparation at different doses and temperatures (control run without the strain). Time course of ethanol production (B) and residual glucose concentration (C) in K. marxianus DSMZ 5422 SSF process with two doses: 20 and 25 AU per gram of starch and at three temperatures: 32, 36 and 40 °C. Letter codes: EtOH, ethanol; GLU, glucose. Values indicate means ± SD from three independent runs.

Figure 3

Time course of (A) dp1 release from raw rice starch by SoAMY-TlGAMY crude preparation at different doses and temperatures (control run without the strain). Time course of ethanol production (B) and residual glucose concentration (C) in K. marxianus DSMZ 5422 SSF process with two doses: 20 and 25 AU per gram of starch and at three temperatures: 32, 36 and 40 °C. Letter codes: EtOH, ethanol; GLU, glucose. Values indicate means ± SD from three independent runs.

Figure 4

Time course of ethanol production and residual glucose concentration in K. marxianus DSMZ 5422 SSF processes. (A) Adjustment of aeration strategy: IS—air provided through a sparger immersed in the culture, HS—air provided from a headspace, IS21—air provided through a sparger immersed in the culture only for the first 21 h of culturing. (B) Comparison of SSF SoAMY-TlGAMY with control processes: using glucose as the carbon source (control G), using commercial STARGEN RSDE (raw starch digesting enzyme) at 20 AU per gram of starch, adopting SHF (separate hydrolysis and fermentation; two-step process with separate stage of 6 h hydrolysis of starch). Letter codes: EtOH, ethanol; GLU, glucose; VC—viable count. Values indicate means ± SD from two independent runs.

Figure 5

(A) Lactic acid production, residual glucose concentration and biomass growth in Lb. plantarum cultures conducted on glucose at different temperatures in two time-points (24 h and 48 h). Letter codes: LA, lactic acid; GLU, glucose. (B) difference in growth characteristics.

Figure 6

Time course of (A) dp1 release at 30 °C by three different doses of SoAMY-TlGAMY preparation; 30, 45 and 60 AU per gram of starch. Time course of (B) lactic acid production and utilization of generated glucose by Lb. plantarum in SSF process conducted on raw starch and cooked starch supplemented with 60 AU per gram of starch. Letter codes: LA, lactic acid; GLU, glucose. Values indicate means ± SD from three independent runs.

Figure 7

Time course of lactic acid production, residual glucose concentration and viable counts in SSF process on cooked starch with Lb. plantarum. Letter codes: LA, lactic acid; GLU, glucose; VC—viable counts. Values indicate means ± SD from two independent runs.