Factors regulating cellulolytic gene expression in filamentous fungi: an overview.

The growing demand for biofuels such as bioethanol has led to the need for identifying alternative feedstock instead of conventional substrates like molasses, etc. Lignocellulosic biomass is a relatively inexpensive feedstock that is available in abundance, however, its conversion to bioethanol involves a multistep process with different unit operations such as size reduction, pretreatment, saccharification, fermentation, distillation, etc. The saccharification or enzymatic hydrolysis of cellulose to glucose involves a complex family of enzymes called cellulases that are usually fungal in origin. Cellulose hydrolysis requires the synergistic action of several classes of enzymes, and achieving the optimum secretion of these simultaneously remains a challenge. The expression of fungal cellulases is controlled by an intricate network of transcription factors and sugar transporters. Several genetic engineering efforts have been undertaken to modulate the expression of cellulolytic genes, as well as their regulators. This review, therefore, focuses on the molecular mechanism of action of these transcription factors and their effect on the expression of cellulases and hemicellulases.

Valorization of xylose-rich hydrolysate from rice straw, an agroresidue, through biosurfactant production by the soil bacterium Serratia nematodiphila.

Biosurfactants, amphiphilic compounds that reduce interfacial tension in oil-aqueous mixtures, are used in the petroleum, pharmaceutical, food, and agriculture industries. Fermentative production of biosurfactants requires expensive sugar or lipid substrates. Lignocellulosic biomass is a relatively cheap and abundant agricultural residue that can be used as an alternative substrate. Currently, several million tonnes of rice and wheat straw are generated globally as agricultural residues, most of which is disposed by open-field burning thereby leading to severe environmental pollution. This study aimed to produce biosurfactants in xylose-rich hydrolysates generated from rice straw. The hydrolysate is also a byproduct of 2G biofuel processes that often goes underutilized. A soil bacterium capable of growing and producing biosurfactants in rice straw hydrolysates, which typically contain growth-inhibitory compounds such as furfural and hydroxymethyl furfural, was isolated. Interestingly, the organism, identified as Serratia nematodiphila, exhibited higher glycolipid formation (4.5 ± 0.6 gL-1) in xylose-rich hydrolysate than in glucose-rich enzymatic hydrolysate (3.1 ± 0.2 gL-1) despite the higher bacterial cell density observed with the latter. The biosurfactants were thermostable and possessed promising emulsifying property and anti-microbial activity against bacteria and yeast. Further optimization of C:N resulted in a 2.8-fold increase in glycolipid production from xylose-rich hydrolysates. This study demonstrates the production of glycolipid biosurfactants from lignocellulosic biomass, a low-cost substrate and offers a plausible strategy for the management of these residues. Further, it also provides insights into the generation of additional high-value compounds in a bioethanol biorefinery to improve its commercial feasibility.

Improvement in ethanol productivity of engineered E. coli strain SSY13 in defined medium via adaptive evolution.

E. coli has the ability to ferment both C5 and C6 sugars and produce mixture of acids along with small amount of ethanol. In our previous study, we reported the construction of an ethanologenic E. coli strain by modulating flux through the endogenous pathways. In the current study, we made further changes in the strain to make the overall process industry friendly; the changes being (1) removal of plasmid, (2) use of low-cost defined medium, and (3) improvement in consumption rate of both C5 and C6 sugars. We first constructed a plasmid-free strain SSY13 and passaged it on AM1-xylose minimal medium plate for 150 days. Further passaging was done for 56 days in liquid AM1 medium containing either glucose or xylose on alternate days. We observed an increase in specific growth rate and carbon utilization rate with increase in passage numbers until 42 days for both glucose and xylose. The 42nd day passaged strain SSK42 fermented 113 g/L xylose in AM1 minimal medium and produced 51.1 g/L ethanol in 72 h at 89% of maximum theoretical yield with ethanol productivity of 1.4 g/L/h during 24-48 h of fermentation. The ethanol titer, yield and productivity were 49, 40 and 36% higher, respectively, for SSK42 as compared to unevolved SSY13 strain.

Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain.

BACKGROUND: Lignocellulosic ethanol production involves major steps such as thermochemical pretreatment of biomass, enzymatic hydrolysis of pre-treated biomass and the fermentation of released sugars into ethanol. At least two different organisms are conventionally utilized for producing cellulolytic enzymes and for ethanol production through fermentation, whereas in the present study a single yeast isolate with the capacity to simultaneously produce cellulases and xylanases and ferment the released sugars into ethanol and xylitol has been described. RESULTS: A yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057 expressed cellulases and xylanases over a wide range of temperatures (32 and 42 °C) and in the presence of different cellulosic substrates [carboxymethylcellulose and wheat straw (WS)]. The studies indicated that the cultivation of yeast at 42 °C in pre-treated hydrolysate containing 0.5 % WS resulted in proportional expression of cellulases (exoglucanases and endoglucanases) at concentrations of 114.1 and 97.8 U g(-1) ds, respectively. A high xylanase activity (689.3 U g(-1) ds) was also exhibited by the yeast under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast occurred within 24 h of incubation. Of the sugars released from biomass after pretreatment, 49 g L(-1) xylose was aerobically converted into 15.8 g L(-1) of xylitol. In addition, 25.4 g L(-1) glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast to obtain an ethanol titer of 7.3 g L(-1). CONCLUSIONS: During the present study, a new strain of C. tropicalis was isolated and found to have potential for consolidated bioprocessing (CBP) applications. The strain could grow in a wide range of process conditions (temperature, pH) and in the presence of lignocellulosic inhibitors such as furfural, HMF and acetic acid. The new yeast produced cellulolytic enzymes over a wide temperature range and in the presence of various cellulosic substrates. The cellulolytic enzymes produced by the yeast were effectively used for the hydrolysis of pretreated biomass. The released sugars, xylose and glucose were, respectively, converted into xylitol and ethanol. The potential shown by the new inhibitor tolerant cellulolytic C. tropicalis to produce ethanol or xylitol is of great industrial significance.

Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli.

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP(+) dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.

Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols.

BACKGROUND: Recent progress in production of various biofuel precursors and molecules, such as fatty acids, alcohols and alka(e)nes, is a significant step forward for replacing the fossil fuels with renewable fuels. A two-step process, where fatty acids from sugars are produced in the first step and then converted to corresponding biofuel molecules in the second step, seems more viable and attractive at this stage. We have engineered an Escherichia coli strain to take care of the second step for converting short chain fatty acids into corresponding alcohols by using butyrate kinase (Buk), phosphotransbutyrylase (Ptb) and aldehyde/alcohol dehydrogenase (AdhE2) from Clostridium acetobutylicum. RESULTS: The engineered E. coli was able to convert butyric acid and other short chain fatty acids of chain length C3 to C7 into corresponding alcohols and the efficiency of conversion varied with different E. coli strain type. Glycerol proved to be a better donor of ATP and electron as compared to glucose for converting butyric acid to butanol. The engineered E. coli was able to tolerate up to 100 mM butyric acid and produced butanol with the conversion rate close to 100% under anaerobic condition. Deletion of native genes, such as fumarate reductase (frdA) and alcohol dehydrogenase (adhE), responsible for side products succinate and ethanol, which act as electron sink and could compete with butyric acid uptake, did not improve the butanol production efficiency. Indigenous acyl-CoA synthetase (fadD) was found to play no role in the conversion of butyric acid to butanol. Engineered E. coli was cultivated in a bioreactor under controlled condition where 60 mM butanol was produced within 24 h of cultivation. A continuous bioreactor with the provision of cell recycling allowed the continuous production of butanol at the average productivity of 7.6 mmol/l/h until 240 h. CONCLUSIONS: E. coli engineered with the pathway from C. acetobutylicum could efficiently convert butyric acid to butanol. Other short chain fatty acids with the chain length of C3 to C7 were also converted to the corresponding alcohols. The ability of engineered strain to convert butyric acid to butanol continuously demonstrates commercial significance of the system.

Fermentation of glycerol and production of valuable chemical and biofuel molecules.

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.

Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.

BACKGROUND: E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products. RESULTS: The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield. CONCLUSIONS: The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.