Abstract
Corynebacterium glutamicum has been safely used in white biotechnology for the last 60 years and the portfolio of new pathways and products is increasing rapidly. Hence, expression vectors play a central role in discovering endogenous gene functions and in establishing heterologous gene expression. In this work, new expression vectors were designed based on two strategies: (i) a library screening of constitutive native and synthetic promoters and (ii) an increase of the plasmid copy number. Both strategies were combined and resulted in a very strong expression and overproduction of the fluorescence protein GfpUV. As a second test case, the improved vector for constitutive expression was used to overexpress the endogenous xylulokinase gene xylB in a synthetic operon with xylose isomerase gene xylA from Xanthomonas campestris. The xylose isomerase activity in crude extracts was increased by about three-fold as compared to that of the parental vector. In terms of application, the improved vector for constitutive xylA and xylB expression was used for production of the N-methylated amino acid sarcosine from monomethylamine, acetate, and xylose. As a consequence, the volumetric productivity of sarcosine production was 50% higher as compared to that of the strain carrying the parental vector.
Highlights
As a consequence, the volumetric productivity of sarcosine production was 50% higher as compared to that of the strain carrying the parental vector
Many of these production strain-engineering efforts rely on gene expression vectors, which represent a powerful tool for metabolic engineering, and for in depth analysis of basic metabolic principles in C. glutamicum that facilitate the development of new metabolic engineering strategies
Precultures of C. glutamicum strains were grown in complex medium Luria broth (LB) (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl) or brain heart infusion (BHI) (37 g/L) supplemented with 50 mL 2% glucose overnight in 500 mL baffled flasks
Summary
C. glutamicum was discovered in the 1960s as a natural L-glutamate producer [1]. Since both its genetic toolbox [2] and its number of heterologous pathways [3,4] have been extended. Besides the sugar polymers starch [16] and cellulose [17], the pentose sugars xylose and arabinose [18,19] that derive from hemicellulose can be used as alternative substrates for a variety of high-value products including the fragrance compound patchoulol [20] and the potential antipsychotic compound sarcosine [11] Many of these production strain-engineering efforts rely on gene expression vectors, which represent a powerful tool for metabolic engineering, and for in depth analysis of basic metabolic principles in C. glutamicum that facilitate the development of new metabolic engineering strategies. In addition to scoring fluorescent reporter gene expression, we applied the gained insight to improve production of the N-methylated amino acid sarcosine from monomethylamine, acetate, and xylose
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