Metabolic Engineering of <i>Candida glabrata</i> for Diacetyl Production

<div><p>In this study, <i>Candida glabrata</i>, an efficient pyruvate-producing strain, was metabolically engineered for the production of the food ingredient diacetyl. A diacetyl biosynthetic pathway was reconstructed based on genetic modifications and medium optimization. The former included (i) channeling carbon flux into the diacetyl biosynthetic pathway by amplification of acetolactate synthase, (ii) elimination of the branched pathway of α-acetolactate by deleting the <i>ILV5</i> gene, and (iii) restriction of diacetyl degradation by deleting the <i>BDH</i> gene. The resultant strain showed an almost 1∶1 co-production of α-acetolactate and diacetyl (0.95 g L⁻¹). Furthermore, addition of Fe³⁺ to the medium enhanced the conversion of α-acetolactate to diacetyl and resulted in a two-fold increase in diacetyl production (2.1 g L⁻¹). In addition, increased carbon flux was further channeled into diacetyl biosynthetic pathway and a titer of 4.7 g L⁻¹ of diacetyl was achieved by altering the vitamin level in the flask culture. Thus, this study illustrates that <i>C. glabrata</i> could be tailored as an attractive platform for enhanced biosynthesis of beneficial products from pyruvate by metabolic engineering strategies.</p></div

Illustration of diacetyl metabolic pathway and pyruvate metabolism in engineered <i>C. glabrata</i>.

<p>The red marks indicate metabolic modifications performed in this study, and the cytosolic ALS encoded by <i>alsS</i> was from <i>B. subtilis</i>. Additional descriptions of the reactions with thiamine and NA as cofactors are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089854#pone.0089854.s005" target="_blank">Table S1</a>.</p

Evaluation of ALS activity and diacetyl production using different ALS enzymes.

<p>The ALS activities were determined in cells at mid-log growth phase of batch-flask fermentation. The mean values of three independent experiments are shown. The error bars indicate the respective standard deviations.</p

Comparison of diacetyl production using different microorganisms in terms of culture medium, engineering strategies, and performance.

<p>Comparison of diacetyl production using different microorganisms in terms of culture medium, engineering strategies, and performance.</p

Comparison of intracellular NADH/NAD⁺ concentrations of strain DA-3 at different vitamin levels.

a<p>A: 0.02 mg L⁻¹ VB1, 8 mg L⁻¹ NA.</p><p>B: 0.08 mg L⁻¹ VB1, 8 mg L⁻¹ NA.</p><p>C: 0.08 mg L⁻¹ VB1, 2 mg L⁻¹ NA. VB₁ and NA represent thiamine and nicotinic acid, respectively.</p

Comparison of DR and BDH activities in the engineered strains and control strain.

<p>The activities of DR and BDH were determined in cells at mid-log growth phase of batch-flask fermentation. The mean values of three independent experiments are shown. The error bars indicate the respective standard deviations.</p

Characteristics of the engineered strain DA-3 in medium containing Cu²⁺, Fe²⁺, and Fe³⁺.

<p>(A) Effects of metal ions on cell growth and ALS activity. (B) The ability of metal ions to transform α-acetolactate to diacetyl in the fermentation process. The present data were obtained after optimizing the titers of the metal ions added with respect to diacetyl production and cell growth.</p

Results of fermentation carried out using strain DA-3 with different concentrations of metal ions and vitamins.

a<p>Control: 0 mM FeCl₃, 0.02 mg L⁻¹ VB₁, 8 mg L⁻¹ NA.</p><p>A: 20 mM FeCl₃, 0.02 mg L⁻¹ VB₁, 8 mg L⁻¹ NA.</p><p>B: 20 mM FeCl₃, 0.08 mg L⁻¹ VB₁, 8 mg L⁻¹ NA.</p><p>C: 20 mM FeCl₃, 0.08 mg L⁻¹ VB₁, 2 mg L⁻¹ NA.</p><p>VB₁ and NA represent thiamine and nicotinic acid, respectively.</p>b<p>The standard deviation for each yield was below 10%.</p

Comparison of fermentation results obtained with metabolically engineered <i>C. glabrata</i> strains and the control strain.

a<p>Concentration of fermentation products (g L⁻¹).</p

<i>Candida glabrata</i> strains and plasmids used in this study.

<p><i>Candida glabrata</i> strains and plasmids used in this study.</p