Engineering Yarrowia lipolytica for the utilization of acid whey.

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 ß-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.

Enhancing hydrogen-dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation.

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO2 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 H2 -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 H2 and CO2 when supplemented with nitrate. Transcriptomic analysis, 13CO2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO2 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 CO2 fixation rate and the production metrics of other chemicals from CO2 and H2 in this host.

Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi).

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) 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.

Genome-scale resources for Thermoanaerobacterium saccharolyticum.

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.

Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum.

BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

Involvement of a bacterial microcompartment in the metabolism of fucose and rhamnose by Clostridium phytofermentans.

BACKGROUND: Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC), organelles composed entirely of proteins. METHODOLOGY AND PRINCIPAL FINDINGS: One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. CONCLUSIONS/SIGNIFICANCE: Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.

Exchange of type II dockerin-containing subunits of the Clostridium thermocellum cellulosome as revealed by SNAP-tags.

Clostridium thermocellum is a thermophilic anaerobic bacterium which efficiently hydrolyzes and metabolizes cellulose to ethanol through the action of its cellulosome, a multiprotein enzymatic complex. A fluorescent protein probe, consisting of a type II dockerin module fused to a SNAP-tag, was developed in order to gain insight into the quaternary configuration of the cellulosome and to investigate the effect of deleting cipA, the protein scaffold on which the cellulosome is built. Fluorescence microscopy suggested that the probe had localized to polycellulosomal protuberances on the cell surface. Surprisingly, fluorescence intensity did not substantially change in the cipA deletion mutants. Sequential labeling experiments suggested that this was a result of bound type II dockerins from CipA being replaced by unbound type II dockerins from the fluorophore-SNAP-XDocII probe. This mechanism of dockerin exchange could represent an efficient means for modifying cellulosome composition.

pB264, a small, mobilizable, temperature sensitive plasmid from Rhodococcus.

BACKGROUND: Gram-positive bacteria of the genus Rhodococcus have shown an extraordinary capacity for metabolizing recalcitrant organic compounds. One hindrance to the full exploitation of Rhodococcus is the dearth of genetic tools available for strain manipulation. To address this issue, we sought to develop a plasmid-based system for genetic manipulation of a variety of Rhodococcus strains. RESULTS: We isolated and sequenced pB264, a 4,970 bp cryptic plasmid from Rhodococcus sp. B264-1 with features of a theta-type replication mechanism. pB264 was nearly identical to pKA22, a previously sequenced but uncharacterized cryptic plasmid. Derivatives of pB264 replicate in a diverse range of Rhodococcus species, showing that this plasmid does not bear the same host range restrictions that have been exhibited by other theta replicating plasmids. Replication or maintenance of pB264 is inhibited at 37 degrees C, making pB264 useful as a suicide vector for genetic manipulation of Rhodococcus. A series of deletions revealed that ca. 1.3 kb from pB264 was sufficient to support replication and stable inheritance of the plasmid. This region includes two open reading frames that encode functions (RepAB) that can support replication of pB264 derivatives in trans. Rhodococcus sp. B264-1 will mobilize pB264 into other Rhodococcus species via conjugation, making it possible to genetically modify bacterial strains that are otherwise difficult to transform. The cis-acting element (oriT) required for conjugal transfer of pB264 resides within a ca. 0.7 kb region that is distinct from the regions responsible for replication. CONCLUSION: Shuttle vectors derived from pB264 will be useful for genetic studies and strain improvement in Rhodococcus, and will also be useful for studying the processes of theta replication and conjugal transfer among actinomycetes.