Consolidated bioprocessing for bioethanol production by metabolically engineered Bacillus subtilis strains

Abstract

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

Metagenomic psychrohalophilic xylanase from camel rumen investigated for bioethanol production from wheat bran using Bacillus subtilis AP

“…B. subtilis is well known for its ability to survive on a variety of substrates. The safety of the bacterium as a GRAS (generally regarded as safe) microorganism as well as its sophisticated systems for production and secretion of enzymes have encouraged the development of ethanologenic B. subtilis AP 23 . This study proved the potential of B. subtilis AP for bioethanol production from wheat bran.…”

“…The Ni–NTA protein purification resin was purchased from Qiagen (Hilden, Germany). The ethanologenic Bacillus subtilis AP [WB600, ldh ::pDH tldh , pHY adh S: pdc Z] was available from a previous study 23 . This stain was deficient in lactate fermentation due to an insertion mutation in the lactate dehydrogenase gene ( ldh ).…”

“…B. subtilis AP was used for bioethanol production from wheat bran. The strain was available from a previous study and just renamed from NS:Z to AP for simplicity 23 . The AP stands for alcohol dehydrogenase and pyruvate decarboxylase.…”

Analysis of Bacterial Diversity in Different Types of Daqu and Fermented Grains From Danquan Distillery

“…Bacillus subtilis can survive in different substrates because of its metabolic diversity and robust systems for the yield and secretion of a diversity of enzymes (Stülke and Hillen, 2000 ; Zhang and Zhang, 2010 ; Gu et al, 2018 ; Banerjee et al, 2019 ). Bacillus subtilis with innate ability to ferment a diversity of carbohydrates from mono-, di-, oligo-, and polysaccharides (such as starch, xylan, galactan, pullulan, arabinan, rhamnogalacturonan, and pectin) seems promising for inexpensive consolidated bioprocessing of bioethanol production using renewable plant biomass and wastes (Maleki et al, 2021 ). In addition, the ethanologenic Bacillus subtilis AP was estimated without and with Xyn-2 for bioethanol production from wheat bran.…”

Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis

“…Therefore, B. subtilis has been considered a biocatalyst for generating valuable compounds from lignocellulosic biomass. Recently, genetic manipulation-based attempts to develop ethanologenic B. subtilis strains have been reported [35,36]. However, it is unclear whether B. subtilis expresses furan aldehyde-reducing AAOR to mediate cellular tolerance to furan aldehydes.…”