Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Abstract

Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles. KEYWORDS: thermostability, keto acid decarboxylase, high-throughput screening, protein design, directed evolution, isobutyl alcohol K eto acid decarboxylases (Kdc) are an important group of

Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature

“…T 1h 50 is the incubationtemperature resulting in ad ecrease to 50 %r esidual activity after 1hincubationo fthe enzyme( for detailed graph, seeFigureS1). [6] All variants, except A290M and A290L, had the same amino acid substitutions as reported by Liao et al [6] Instead of purifying each variant, stepwise SSM was performed. The value of T 1h 50 is related to that of T m ,b ut the two values are not necessarily identical, because the former is time-dependent and describest he kinetic stability, and the latter is at hermodynamic descriptor of thermostability.…”

“…Effect of single amino acid exchanges on enhanced T 1h 50 of KdcA. [6] It wasl ogical to assume that thesea mino acid residues would have as imilar effect on KdcA, since both have similar protein sequence identities (80 %). T 1h 50 is the incubationtemperature resulting in ad ecrease to 50 %r esidual activity after 1hincubationo fthe enzyme( for detailed graph, seeFigureS1).…”

“…Approachesc onsidereda re presented on the right. [6] After as econd screening, severalv ariantsw ith increased thermostability were identified: Q34H, A290M, A290L, A290V,a nd T386P.N oi mprovedv ariant was detected in the L130 library. of 64 8Cc orresponds to a3 8Ci ncreaser elative to the WT enzyme ( Table 1).…”

“…[3] In an effort to avoid this in vivo toxicity of isobutanol, in vitro alcohol production has been reported to withstand iso-butanol concentrations up to 4%. [6] Isobutanol production in the thermophilicb acterium Geobacillus thermoglucosidasius has been reported recently;h owever,t he yield was much lower than in the process performed with E. coli. Higher temperatures avoid the risk of contamination and may allow biomass pretreatmenta nd alcohol fermentation to be performed simultaneously in one pot while speeding up the production process and loweringc osts.…”

“…[7] One explanation suggestst hat one of the key enzymes,n amely,t he branched-chain keto acid decarboxylase from Lactoccocus lactis (KivD), which catalyzes the decarboxylation of 3-methyl-2-oxobutanoic acid (ketoisovalerate) to 2-methylpropanal (isobutanal), is not thermostable. [6] Sulfolobus acidocaldarius (Saci)i sathermoacidophilic archaea that is ablet og row and prosper at 75 8Ca nd at pH 2-3. [5] Recent work on KivDb yd irected evolution (epPCR) and computational design wasa ble to increase the temperature at which 50 %o fa ctivity remains after one-hour incubation T 1h 50 from 56 to 60 8C(LLM4 mutant).…”