Abstract
Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).
Highlights
IntroductionBiorefinery processes have been developed to establish a sustainable alternative to produce chemicals, polymers, and fuels, and they currently rely on petroleum-based processes [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].Development of recombinant microorganisms capable of converting a broader range of renewable biomass feedstock into bio-based chemicals, with properties comparable to those of conventional petrochemical products, have been extensively studied [18,19,20,21,22,23,24,25,26,27]
To investigate the tunability of protein expression using Pvgb in recombinant C. glutamicum, several expression vector systems based on pCES208 plasmids were constructed
green fluorescent protein (GFP) was expressed in C. glutamicum under the control of increasing repeats of Pvgb (Pvgb, Pvgb4, Pvgb8 )
Summary
Biorefinery processes have been developed to establish a sustainable alternative to produce chemicals, polymers, and fuels, and they currently rely on petroleum-based processes [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].Development of recombinant microorganisms capable of converting a broader range of renewable biomass feedstock into bio-based chemicals, with properties comparable to those of conventional petrochemical products, have been extensively studied [18,19,20,21,22,23,24,25,26,27]. Production of amino acid-derived products such as 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) from glucose using recombinant E. coli is limited, due to its inability to accumulate important precursors such as L-lysine and L-glutamate [24,27]. In this regard, Corynebacterium glutamicum, which is a generally recognized as safe (GRAS) strain, is an excellent workhorse to develop because of its innate ability to produce large amounts of amino acids such as L-lysine and L-glutamate [1,4,5]. Several studies have demonstrated that C. glutamicum can produce high levels of bio-based nylon monomers such putrescine, succinic acid [4], GABA [27], cadaverine [28,29], 5-AVA and glutaric acid, [30]
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