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Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted β-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO 2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H 2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H 2 and CO 2 when supplemented with nitrate. Transcriptomic analysis, 13 CO 2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO 2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products. This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO 2 fixation rate and the production metrics of other chemicals from CO 2 and H 2 in this host., (© 2018 Wiley Periodicals, Inc.)

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO 2 , H 2 ) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii., (Copyright © 2018. Published by Elsevier Inc.)

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation., Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results., Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.