Directed evolution of promising engineered strains to improve their production capacity has received widespread attention in recent years. This study aims to develop a robust C. tyrobutyricum chassis which has an alternative non-oxidative glycolysis (NOG) pathway to EMP pathway and can withstand possible growth side effects of EMP modification during genomic directed evolution for enhanced or expanded synthesis capacity of bioproducts. We integrated a non-oxidative glycolysis (NOG) pathway to EMP pathway in C. tyrobutyricum by introducing F/Xpk gene. Plasmids using two F/Xpk genes and three different native promoters, Pthl, Ptkt and Pfba, were constructed and transferred into C. tyrobutyricum L319. By analyzing the growth and yields of butyric acid and acetic acid of the strain, we found that F/Xpk gene derived from C. acetobutylicum and Ptkt promoter was the best combination for constructing NOG pathway in C. tyrobutyricum, and xylose was the best carbon source for the strain.