Construction of a strain of Lactobacillus casei that co-utilizes glucose and maltose for industrial production of L-lactic acid.

摘要

Lactic acid and its derivatives have numerous industrial uses and demand for L(+) lactic acid is rapidly growing. Lactic acid can be manufactured by fermentation or via chemical synthesis. Fermentation is the preferred method, due to lower environmental concerns and the ability to selectively produce the L(+) isomer. Corn steep liquor (CSL), due to its low cost and nutrient quality, is the preferred fermentation substrate. However, the presence of maltose in CSL results in problems during purification, thereby greatly increasing the costs. Lactobacillus casei, the fermentation organism of choice, does not utilize the maltose present in CSL. The objective of this research was to construct a Lb. casei strain that utilizes glucose and maltose concurrently. This strain would have enhanced industrial utility in the production of L-lactate by fermentation.; In this study, the pY0005 encoded maltose catabolite gene cluster from Lactobacillus sakei was sequenced and characterized. The nucleotide sequence identified of five open reading frames (ORF) putatively involved in maltose catabolism: transcriptional regulator (malR), maltose permease (malK), maltose phosphorylase ( malP), galactose mutarotase (galM and beta-phosphoglucomutase (pgmA). Additionally, a putative catabolite responsive element (cre) was identified in promoter region of malK. Three genes, malR, galM, and pgmA, were determined to be monocistronic and expressed in the presence of both glucose and maltose. Two genes, malK and malP, were determined to be co-transcribed and were not expressed in the presence of glucose. MalR was determined to be repressor of the malK-malP transcript in the presence of glucose. The cre site and malK-malP promoter were replaced with the L-ldh promoter from Lb. casei. This construct was utilized to examine carbohydrate utilization and transport in Lb. casei ATCC334 and Lb. sakei ML291. We determined that putative cre sequence was not required for glucose catabolite repression in Lb. sakei ML291. Rather, glucose catabolite repression is due to inducer exclusion of maltose transport in Lb. sakei ML291. However, glucose did not repress maltose utilization in Lb. casei ATCC334. Therefore, Lb. casei constructs containing the pYC005 maltose cluster may have utility in the production of L(+) lactic acid by fermentation.