title: Construction of a constitutively expressed homo-fermentative pathway in Lactobacillus brevis.
abstract: Lactobacillus brevis is a promising lactic acid producing strain that simultaneously utilizes glucose and xylose from lignocellulosic hydrolysate without carbon catabolic repression and inhibition. The production of by-products acetic acid and ethanol has been the major drawback of this strain. Two genes, pfkA (fructose-6-phosphate kinase [PFK]) and fbaA (fructose-1,6-biphosphate aldolase [FBA]), that encode the key enzymes of the EMP/glycolytic pathway from Lactobacillus rhamnosus, were fused to the downstream of the strong promoter P32 and expressed in L. brevis s3f4 as a strategy to minimize the formation of by-products. By expressing the two enzymes, a homo-fermentative pathway for lactic acid production was constructed. The lactic acid yields achieved from glucose in the transformants were 1.12 and 1.16 mol/mol, which is higher than that of the native strain (0.74 mol/mol). However, the lactic acid yield from xylose in the transformants stayed the same as that of the native strain. Enzyme assay indicated that the activity of the foreign protein FBA in the transformants was much higher than that of the native strains, but was ten times lower than that in L. rhamnosus. This result was consistent with the metabolic flux analysis, which indicated that the conversion efficiency of the expressed PFK and FBA was somewhat low. Less than 20 % of the carbons accumulated in the form of fructose-6-phosphate were converted into glyceraldehyde-3-phosphate (GAP) by the expressed PFK and FBA. Metabolic flux analysis also indicated that the enzyme phosphoketolase (XPK) played an important role in splitting the carbon flow from the pentose phosphate pathway to the phosphoketolase pathway. This study suggested that the lactic acid yield of L. brevis could be improved by constructing a homo-fermentative pathway.
authors.affiliation: Tianjin Key Laboratory for Industrial BioSystem and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xiqidao, Airport Economic Park, Tianjin, 300308, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.forename: Ronglin
authors.initials: R
authors.lastname: He
authors.forename: Lijuan
authors.initials: L
authors.lastname: Ma
authors.forename: Wendi
authors.initials: W
authors.lastname: Jia
authors.forename: Demao
authors.initials: D
authors.lastname: Li
authors.forename: Shulin
authors.initials: S
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism*; D052196:Levilactobacillus brevis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D052201:Lacticaseibacillus rhamnosus / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019344:Lactic Acid; D014994:Xylose; D010732:Phosphofructokinase-1; D005634:Fructose-Bisphosphate Aldolase; D005947:Glucose