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1. Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

   https://doi.org/10.1002/elsc.202200054  

   Moxley, W. Chris ; Brown, Rachel E. ; Eiteman, Mark A. ( January 2023 , Engineering in Life Sciences)

   Several chromosomally expressed AceE variants were constructed inEscherichia coli ΔldhA ΔpoxB ΔppsAand compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of thebudAandbudBgenes fromEnterobacter cloacae ssp. dissolvens. The best acetoin‐producing strains were subsequently studied in controlled batch culture at the one‐liter scale. PDH variant strains attained up to four‐fold greater acetoin than the strain expressing the wild‐type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate‐derived products, acetoin (38.5 g/L) and 2R,3R‐butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.
2. Engineering and comparison of non‐natural pathways for microbial phenol production

   https://doi.org/10.1002/bit.25942  

   Thompson, Brian ; Machas, Michael ; Nielsen, David R. ( February 2016 , Biotechnology and Bioengineering)

   The non‐renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non‐natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediatep‐hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL‐dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745–1754. © 2016 Wiley Periodicals, Inc.
3. Engineered citrate synthase improves citramalic acid generation in Escherichia coli

   https://doi.org/10.1002/bit.27450  

   Wu, Xianghao ; Tovilla‐Coutiño, D. Brisbane ; Eiteman, Mark A. ( June 2020 , Biotechnology and Bioengineering)

   The microbial product citramalic acid (citramalate) serves as a five‐carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized inEscherichia colidirectly by the condensation of pyruvate and acetyl‐CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl‐CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in thegltAgene coding citrate synthase prevents acetyl‐CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study theE. colicitrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl‐CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed‐batch process using MEC626/pZE12‐cimAharboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can bemore »used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.

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4. The XylR variant (R121C and P363S) releases arabinose‐induced catabolite repression on xylose fermentation and enhances coutilization of lignocellulosic sugar mixtures

   https://doi.org/10.1002/bit.27144  

   Martinez, Rodrigo ; Flores, Andrew D. ; Dufault, Matthew E. ; Wang, Xuan ( August 2019 , Biotechnology and Bioengineering)

   Microbial production of fuels and chemicals from lignocellulosic biomass provides a promising alternative to conventional petroleum‐derived routes. However, the heterogeneous sugar composition of lignocellulose prevents efficient microbial sugar co‐fermentation due to carbon catabolite repression, which negatively affects production metrics. We previously discovered that a mutant copy of the transcriptional regulator XylR (P363S and R121C; denoted as XylR*) inEscherichia colihas a higher DNA‐binding affinity than wild‐type XylR, leading to a stronger activation of thed‐xylose catabolic genes and a release from glucose‐induced repression on xylose fermentation. Here, we showed that XylR* also releasesl‐arabinose‐induced repression on xylose fermentation through altered transcriptional control, enhancing co‐fermentation of arabinose–xylose sugar mixtures in wild‐typeE. coli. IntegratingxylR*into an ethanologenicE. coliresulted in the coutilization of 96% of the provided glucose–xylose–arabinose mixtures (120 g/L total sugars supplied) with an ethanol yield higher than 90% of the theoretical maximum by simple batch fermentations.
5. Uncovering Lasonolide A Biosynthesis Using Genome-Resolved Metagenomics

   https://doi.org/10.1128/mbio.01524-22  

   Uppal, Siddharth ; Metz, Jackie L. ; Xavier, René K. ; Nepal, Keshav Kumar ; Xu, Dongbo ; Wang, Guojun ; Kwan, Jason C. ( October 2022 , mBio)

   Ravel, Jacques (Ed.)

   ABSTRACT Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named “ Candidatus Thermopylae lasonolidus,” only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A ( las ) biosynthetic gene cluster (BGC) was identified as a trans -acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein inmore »one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. IMPORTANCE While sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small quantities by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations, which causes premature chromosome condensation, blebbing, cell contraction, and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anticancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing, identified genes likely responsible for LSA synthesis in an uncultured bacterium, and assembled the symbiont’s genome. These insights provide future opportunities for heterologous expression and cultivation efforts that may minimize LSA’s supply problem.« less