Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate

Background Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. Results The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. Conclusions In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Antimicrobial Applications of Rhamnolipid Biosurfactant Produced from Achromobacter sp. (PS1) Isolate Using Lignocellulosic Hydrolysate

Heterogeneous mixture of partially purified rhamnolipid (RL) produced from Achromobacter sp. (PS1) using lignocellulosic rice straw (RS) sugar hydrolysate medium revealed six different congeners- Rha- C10-C10, Rha-C8-C10/Rha-C10-C8, Rha- C12-C10 / Rha- C10-C12, referring mono-rhamnolipids amounting to total 68.23 % and Rha-Rha-C10-C10, Rha-Rha-C8-C10/Rha-Rha-C10-C8, Rha-Rha-C10-C12/Rha-Rha-C12-C10, referring di-rhamnolipids amounting to 31.73 %, with Mono to Di- RL in the ratio of 2.1:1. This mixture's antimicrobial action containing more mono-rhamnolipids analyzed using broth macro-dilution method exhibited a broad-spectrum antibacterial activity showing ≥ 90 % growth inhibition of both Gram-positive and Gram-negative pathogenic bacteria at MIC ranging from 1.25 mg/mL to 10 mg/mL of total rhamnolipids. This might be due to the more hydrophobic character of mono-rhamnolipids containing a single rhamnosyl group and showing high surface activities. On the other hand, the non-antifungal activity may be attributed to the lower percentage of di-rhamnolipids in the partially purified mixture.

Efficient lactic acid production from dilute acid-pretreated lignocellulosic biomass by a synthetic consortium of engineered Pseudomonas putida and Bacillus coagulans

Background Lignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production of a range of biochemicals, and pretreatment is generally regarded as indispensable for its biorefinery. However, various inhibitors that severely hinder the growth and fermentation of microorganisms are inevitably produced during the pretreatment of lignocellulose. Presently, there are few reports on a single microorganism that can detoxify or tolerate toxic mixtures of pretreated lignocellulose hydrolysate while effectively transforming sugar components into valuable compounds. Alternatively, microbial coculture provides a simpler and more efficacious way to realize this goal by distributing metabolic functions among different specialized strains. Results In this study, a novel synthetic microbial consortium, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from highly toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of the sugar metabolism pathway was unable to consume the major fermentable sugars of lignocellulosic hydrolysate but exhibited great tolerance to 10 g/L sodium acetate, 5 g/L levulinic acid, 10 mM furfural and HMF as well as 2 g/L monophenol compound. In addition, the engineered strain rapidly removed diverse inhibitors of real hydrolysate. The degradation rate of organic acids (acetate, levulinic acid) and the conversion rate of furan aldehyde were both 100%, and the removal rate of most monoaromatic compounds remained at approximately 90%. With detoxification using engineered P. putida for 24 h, the 30% (v/v) hydrolysate was fermented to 35.8 g/L lactic acid by B. coagulans with a lactic acid yield of 0.8 g/g total sugars. Compared with that of the single culture of B. coagulans without lactic acid production, the fermentation performance of microbial coculture was significantly improved. Conclusions The microbial coculture system constructed in this study demonstrated the strong potential of the process for the biosynthesis of valuable products from lignocellulosic hydrolysates containing high concentrations of complex inhibitors by specifically recruiting consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for the bioconversion of lignocellulosic biomass to other value-added biochemicals.

Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate

Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase.

RNA sequencing reveals metabolic and regulatory changes leading to more robust fermentation performance during short-term adaptation of Saccharomyces cerevisiae to lignocellulosic inhibitors

Background The limited tolerance of Saccharomyces cerevisiae to inhibitors is a major challenge in second-generation bioethanol production, and our understanding of the molecular mechanisms providing tolerance to inhibitor-rich lignocellulosic hydrolysates is incomplete. Short-term adaptation of the yeast in the presence of dilute hydrolysate can improve its robustness and productivity during subsequent fermentation. Results We utilized RNA sequencing to investigate differential gene expression in the industrial yeast strain CR01 during short-term adaptation, mimicking industrial conditions for cell propagation. In this first transcriptomic study of short-term adaption of S. cerevisiae to lignocellulosic hydrolysate, we found that cultures respond by fine-tuned up- and down-regulation of a subset of general stress response genes. Furthermore, time-resolved RNA sequencing allowed for identification of genes that were differentially expressed at 2 or more sampling points, revealing the importance of oxidative stress response, thiamin and biotin biosynthesis. furan-aldehyde reductases and specific drug:H+ antiporters, as well as the down-regulation of certain transporter genes. Conclusions These findings provide a better understanding of the molecular mechanisms governing short-term adaptation of S. cerevisiae to lignocellulosic hydrolysate, and suggest new genetic targets for improving fermentation robustness.

Efficient Lactic Acid Production from Dilute Acid Pretreated Lignocellulosic Biomass by a Synthetic Consortia of Engineered Pseudomonas Putida and Bacillus Coagulans

Background Lignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production a range of biochemicals, and a pretreatment is generally regarded to be indispensable for its bio-refinery. Nevertheless, various inhibitors that severely hindered the growth and fermentation of microorganisms were produced inevitably during the pretreatment of lignocellulose. Presently, a single microorganism that can tolerate toxic mixtures of pretreatment hydrolysate while effectively transforming sugar components into valuable compound is less well reported. Alternatively, microbial co-culture provides a simpler and more efficacious way to realize this goal via distributing metabolic tasks among proper strains. Results In this study, a novel synthetic microbial consortia, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from high-toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of sugar metabolism pathway was suggested to be unable to consume the major fermentable sugars of lignocellulosic hydrolysate, but can rapidly remove inhibitors in hydrolysate. With detoxification using engineered P. putida for 24 h, the pretreated hydrolysate was fermented into 35.8 g/L of lactic acid by B. coagulans with a yield of 90%. The fermentation performance of microbial co-culture was significantly improved than that single culture of B. coagulans without lactic acid production. Conclusions The microbial coculture system constructed by this study demonstrated strong potential of the process for biosynthesis of valuable product from lignocellulosic hydrolysate containing high concentration of complex inhibitors by specifically recruited consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for bioconversion of lignocellulosic biomass to other value-added biochemicals.