Consolidated bioprocessing for bioethanol production by metabolically engineered Bacillus subtilis strains

Fatemeh Maleki,Narges Zolfaghari,Hossein Shahbani Zahiri,Sarah Rajaei,Mohammad Changizian,Kambiz Akbari Noghabi

doi:10.1038/s41598-021-92627-9

Fatemeh Maleki, Narges Zolfaghari + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-92627-9

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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.

Highlights

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel
The only successful attempt to develop an ethanologenic strain of B. subtilis has been reported by Romero et al.[6]. They managed to create the strain by engineering an exogenous ethanol pathway using heterologous expression of Z. mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase
A positive point of B. subtilis compared to natural ethanologenic strains such as S. cerevisiae is that the bacterium can utilize polysaccharides, and it might be used in Consolidated bioprocessing (CBP) systems for affordable bioethanol production from plant biomass without the need for enzymatic pretreatments

Summary

Introduction

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. They managed to create the strain by engineering an exogenous ethanol pathway using heterologous expression of Z. mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) They had to knock out the native genes encoding lactate dehydrogenase (ldh) and acetolactate synthase (alsS) to obstruct lactate and 2,3-butanediol production, respectively, as major fermentation products of B. subtilis. A positive point of B. subtilis compared to natural ethanologenic strains such as S. cerevisiae is that the bacterium can utilize polysaccharides, and it might be used in CBP systems for affordable bioethanol production from plant biomass without the need for enzymatic pretreatments. A plausible solution to this challenge is to combine the saccharification and fermentation into a single step by CBP in which a microorganism is responsible to catalyze the whole process With regard to this point, the current study was conducted to evaluate the potential of B. subtilis for bioethanol production from untreated potatoes in a consolidated bioprocess (Fig. 1). The resulting strains were investigated for ethanol production on glucose as well as for CBP ethanol production on potatoes

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