Specific activity of enzymes of the ethylmalonyl-CoA pathway and their regulation.

<p>Enzymes: PhaA, β-ketothiolase; PhaB, acetoacetyl-CoA reductase; CroR, crotonase; Ccr, crotonyl-CoA carboxylase/reductase; Epi, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; Ecm, ethylmalonyl-CoA mutase; Msd, methylsuccinyl-CoA dehydrogenase; Mcd, mesaconyl-CoA hydratase; Mcl1, malyl-CoA/β-methylmalyl-CoA lyase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.</p

Specific activity of enzymes of the C₂ specific steps of the ethylmalonyl-CoA pathway and their regulation.

<p>Enzymes: Pcc, propionyl-CoA carboxylase; Epi, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; Mcm, methylmalonyl-CoA mutase; Mcl2, malyl-CoA thioesterase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.</p

Specific activity of enzymes of the serine cycle and their regulation.

<p>Enzymes: Mox, methanol dehydrogenase; GlyA, serine hydroxymethyl transferase; Sga, serine-glyoxylate aminotransferase; Hpr, hydroxypyruvate reductase; Gck, glycerate kinase; Eno, enolase; Ppc, phosphoenolpyruvate carboxylase; Mdh, malate dehydrogenase; Mtk, malate-CoA ligase; Mcl1, malyl-CoA/β-methylmalyl-CoA lyase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.</p

Scheme of C₁ metabolism of the methylotroph <i>Methylobacterium extorquens</i> AM1.

<p>The bacterium oxidizes methanol to formaldehyde that is condensed with a tetrahydromethanopterin and further oxidized to formate. Formate reacts with tetrahydropterin and formyl-tetrahydrofolate is further converted to methylenetetrahydrofolate (part 1 of metabolism). The serine cycle is used for the assimilation of formaldehyde plus bicarbonate (part 2). Acetyl-CoA assimilation and conversion to glyoxylate proceeds <i>via</i> the ethylmalonyl-CoA pathway (part 3). The main biosynthetic outputs from these pathways are indicated. Enzymes: 1, serine hydroxymethyl transferase; 2, serine-glyoxylate aminotransferase; 3, hydroxypyruvate reductase; 4, glycerate kinase; 5, enolase; 6, phosphoenolpyruvate carboxylase; 7, malate dehydrogenase; 8, malate-CoA ligase (malate thiokinase); 9, L-malyl-CoA/β-methylmalyl-CoA lyase; 10, β-ketothiolase; 11, acetoacetyl-CoA reductase; 12, crotonase (<i>R</i>-specific); 13, crotonyl-CoA carboxylase reductase; 14, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; 15, ethylmalonyl-CoA mutase; 16, methylsuccinyl-CoA dehydrogenase; 17, mesaconyl-CoA hydratase; 18, propionyl-CoA carboxylase; 19, methylmalonyl-CoA mutase; 20, methanol dehydrogenase. PHB, polyhydroxybutyrate, Q, quinone.</p

Postulated limiting steps in methanol assimilation in <i>M. extorquens</i> AM1.

<p>The grey dashed lines indicate the calculated minimal value for the specific activity of enzymes, which is required to account for the observed generation time of 3 hours on methanol.</p

Specific activities of methanol dehydrogenase, of enzymes of the serine cycle and of the ethylmalonyl-CoA pathway in extracts of <i>M. extorquens</i> cells grown on methanol, acetate or succinate.

<p>*The values shown are means ± standard deviations of results from at least three independent measurements.</p>#<p>n. d. = not determined.</p>1<p>cleavage of malyl-CoA;</p>2<p>cleavage of β-methylmalyl-CoA.</p

Multiple Functions of the Type II Thioesterase Associated with the Phoslactomycin Polyketide Synthase

Polyketide synthases (PKSs) are molecular assembly lines that condense basic chemical building blocks for the production of structurally diverse polyketides. Many PKS biosynthetic gene clusters contain a gene encoding for a type II thioesterase (TEII). It is believed that TEIIs exert a proofreading function and restore or increase the productivity of PKSs by removing aberrant modifications on the acyl-carrier proteins (ACPs) of the PKS assembly line. Yet biochemical evidence is still sparse. Here, we investigated the function of PnG, the TEII of the phoslactomycin PKS (Pn PKS), in the context of its cognate assembly line in vitro. Biochemical analysis revealed that PnG preferentially hydrolyzes alkyl-ACPs over (alkyl)malonyl-ACPs by up to three orders of magnitude, supporting a proofreading role of the enzyme. We further demonstrate that PnG increases the in vitro production of different native and non-native tetra-, penta-, and hexaketide derivatives of phoslactomycin by more than one order of magnitude and show that these effects are caused by the initial clearing of the Pn PKS, as well as proofreading of the active assembly line. Finally, we demonstrate that PnG is able to release intermediate but notably also terminal polyketides from the Pn PKS. This allows PnG to functionally replace and overcome the terminal TEI activity of chimeric in vitro Pn PKS systems, as showcased with a phoslactomycin hexaketide system. Altogether, our experiments provide detailed insights into the molecular mechanisms and the multiple functions of PnG in its native context, as well as their potential use in future applications

A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in <i>Methylobacterium extorquens</i> AM1 and Other Alphaproteobacteria

The discipline of synthetic biology requires standardized tools and genetic elements to construct novel functionalities in microorganisms; yet, many model systems still lack such tools. Here, we describe a novel set of vectors that allows the convenient construction of synthetic operons in Methylobacterium extorquens AM1, an important alphaproteobacterial model organism for methylotrophy and a promising platform organism for methanol-based biotechnology. In addition, we provide a set of constitutive alphaproteobacterial promoters of different strengths that were characterized in detail by two approaches: on the single-cell scale and on the cell population level. Finally, we describe a straightforward strategy to deliver synthetic constructs to the genome of M. extorquens AM1 and other Alphaproteobacteria. This study defines a new standard to systematically characterize genetic parts for their use in M. extorquens AM1 by using single-cell fluorescence microscopy and opens the toolbox for synthetic biological applications in M. extorquens AM1 and other alphaproteobacterial model systems

Replacing the Ethylmalonyl-CoA Pathway with the Glyoxylate Shunt Provides Metabolic Flexibility in the Central Carbon Metabolism of <i>Methylobacterium extorquens</i> AM1

The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium <i>Methylobacterium extorquens</i> AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in <i>M. extorquens</i> AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in <i>M. extorquens</i> AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future

A Family of Single Copy <i>repABC</i>-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium <i>Sinorhizobium meliloti</i>

A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six <i>repABC</i>-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the <i>repABC</i> region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and <i>Agrobacterium</i>-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of <i>repABC</i> regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in <i>Sinorhizobium meliloti</i>, related members of the <i>Rhizobiales</i>, and <i>Escherichia coli</i>. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) <i>repABC</i> cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In <i>S. meliloti</i>, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in <i>S. meliloti</i>, suggesting combinability of multiple compatible pABC plasmids. We further devised an <i>in vivo</i> cloning strategy involving Cre/<i>lox</i>-mediated translocation of large DNA fragments to an autonomously replicating <i>repABC</i>-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or <i>E. coli</i>