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d-Allulose is considered an ideal alternative to sucrose and has shown tremendous application potential in many fields. Recently, most efforts on production of d-allulose have focused on in vitro enzyme-catalyzed epimerization of cheap hexoses. Here, we proposed an approach to efficiently produce d-allulose through fermentation using metabolically engineered Escherichia coli JM109 (DE3), in which a SecY (ΔP) channel and a d-allulose 3-epimerase (DPEase) were co-expressed, ensuring that d-fructose could be transported in its nonphosphorylated form and then converted into d-allulose by cells. Further deletion of fruA, manXYZ, mak, galE, and fruK and the use of Ni2+ in a medium limited the carbon flux flowing into the byproduct-generating pathways and the Embden-Meyerhof-Parnas (EMP) pathway, achieving a ≈ 0.95 g/g yield of d-allulose on d-fructose using E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 μM Ni2+. In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.

Consolidated bioprocessing (CBP) of cellulose mixed with fermentable sugar(s) is considered as a promising alternative to the use of cellulose as sole substrate for bioethanol production. Our research metabolically engineered Saccharomyces cerevisiae to allow for the co-conversion of cellulose and either sucrose or xylose to bioethanol. Constitutive promoter substitution and xylose metabolic pathway integration were carried out in a strain previously modified to express both bifunctional minicellulosomes by galactose induction and a cellodextrin pathway. Strain EBY101-CC, engineered for the co-fermentation of cellulose and sucrose, produced 4.3 g/L ethanol from 10 g/L carboxymethyl cellulose (CMC) and batch-fed sucrose with an ethanol yield of 0.43 g/g of total sugars. Strains modified for co-fermentation of xylose and cellulose, EBY101-X5CC and EBY101-X5CP were able to produce 2.9 g/L cellulosic ethanol from 8.0 g/L CMC and 1.2 g/L from 3.2 g/L phosphoric acid-swollen cellulose (PASC), respectively, when...

1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD+-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD+-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or...