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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.)

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg -1 ) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

In previous work, we constructed a recombinant Bacillus subtilis strain for microbial production of N-acetylglucosamine (GlcNAc), which has applications in nutraceuticals and pharmaceuticals. In this work, we improve GlcNAc production through modular engineering of B. subtilis. Specifically, the GlcNAc synthesis-related metabolic network in B. subtilis was divided into three modules-GlcNAc synthesis, glycolysis, and peptidoglycan synthesis. First, two-promoter systems with different promoter types and strengths were used for combinatorial assembly of expression cassettes of glmS (encoding GlcN-6-phosphate synthase) and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) at transcriptional levels in the GlcNAc synthesis module, resulting in a 32.4% increase in GlcNAc titer (from 1.85g/L to 2.45g/L) in shake flasks. In addition, lactate and acetate synthesis were blocked by knockout of ldh (encoding lactate dehydrogenase) and pta (encoding phosphotransacetylase), leading to a 44.9% increase in GlcNAc production (from 2.45g/L to 3.55g/L) in shake flasks. Then, various strengths of the glycolysis and peptidoglycan synthesis modules were constructed by repressing the expression of pfk (encoding 6-phosphofructokinase) and glmM (encoding phosphoglucosamine mutase) via the expression of various combinations of synthetic small regulatory RNAs and Hfq protein. Next, GlcNAc, glycolysis, and peptidoglycan synthesis modules with various strengths were assembled and optimized via a module engineering approach, and the GlcNAc titer was improved to 8.30g/L from 3.55g/L in shake flasks. Finally, the GlcNAc titer was further increased to 31.65g/L, which was 3.8-fold that in the shake flask, in a 3-L fed-batch bioreactor. This work significantly enhanced GlcNAc production through modular pathway engineering of B. subtilis, and the engineering strategies used herein may be useful for the construction of versatile B. subtilis cell factories for the production of other industrially important chemicals., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Most of the methane produced in nature derives from the methyl group of acetate, the major end product of anaerobes decomposing complex plant material. The acetate is derived from the metabolic intermediate acetyl-CoA via the combined activities of phosphotransacetylase and acetate kinase. In Methanosarcina species, the enzymes function in the reverse direction to activate acetate to acetyl-CoA prior to cleavage into a methyl and carbonyl group of which the latter is oxidized providing electrons for reduction of the former to methane. Thus, phosphotransacetylase and acetate kinase have a central role in the conversion of complex organic matter to methane by anaerobic microbial food chains. Both enzymes have been purified from Methanosarcina thermophila and characterized. Both enzymes from M. thermophila have also been produced in Escherichia coli permitting crystal structures and amino acid variants, the kinetic and biochemical studies of which have lead to proposals for catalytic mechanisms. The high identity of both enzymes to paralogs in the domain Bacteria suggests ancient origins and common mechanisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)