Metabolic engineering of Thermoanaerobacterium aotearoense SCUT27 for glucose and cellobiose co-utilization by identification and overexpression of the endogenous cellobiose operon

Abstract Cellobiose is abundant in partial hydrolysis of lignocellulosic hydrolysates for the weak activity of β-glucosidase, the rate-limiting step in the enzymatic hydrolysis. Thermoanaerobacterium aotearoense SCUT27 termed SCUT27 has broad substrate spectrums and can co-utilize glucose and xylose, xylose and cellobiose. However, SCUT27 can’t co-utilize glucose and cellobiose due to carbon catabolite repression (CCR), which couldn’t be eliminated after ccpA knockout. The ability of co-utilizing glucose and cellobiose is a desirable feature of SCUT27 for industrial application. Four operons related to cellobiose utilization had been explored in SCUT27, named as Cel1, Cel2, Cel3 and Cel4. RT-PCR results showed that Cel2 operon is the only intact operon including integral cellobiose PTS transport elements and glycoside hydrolase, which is very important for cellobiose utilization. The transcription of Cel2 was regulated by σ54-dependent transcription activator CelRcel2 (V518_2450). With sigma factor 54 (σ54) deletion, the transcription strength of Cel2 decreased significantly. By replacing the σ54-dependent promoter with the strong promoter of gene adhE (V518_0444), gene cluster bglpcel2EIIABCcel2 (V518_2444−2449) could be easily overexpressed and SCUT27/ΔcelRcel2/padhE obtained the ability to co-utilize glucose and cellobiose, which was a potential candidate for ethanol or lactic acid fermentation from partial hydrolysis of lignocellulosic hydrolysates.