Bacterial strains and plasmids.

<p><b>Abbreviations:</b> Ap^R, ampicillin resistance; Cm^R, chloramphenicol resistance; MLS^R, macrolide, lincosamide, and streptogramin B resistance; Tet^R, tetracycline resistance; Ф<i>3tI</i>, Ф3TI methyltransferase gene of <i>Bacillus subtilis</i> phage Ф3TI. Pthl, thiolase gene promoter (Pthl) from <i>C. acetobutylicum;</i> DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; NEB, New England Biolabs Beverly, MA.</p

Construction of CAC1502 disrupted mutant <i>C. acetobutylicum</i> SMB009.

<p><b>A.</b> Schematic show of the position of CAC1502 and the expected disrupted CAC1502 in the chromosome. 86-CAC1502-1 and 87-CAC1502-2 are the primers for generation of CAC1502 probe. <b>B.</b> Identification of the mutant SMB009 by PCR using primers 86-CAC1502-1 and 87-CAC1502-2 flanking the insertion site. <b>C.</b> Southern blot confirmation of intron insertion into CAC1502 using the intron probe and the CAC1502 specific probe.</p

Detection of Cac824I activity in <i>C. acetobutylicum</i> mutant SMB009 using methylated or unmethylated pMTL007 as DNA substrate.

<p><b>A.</b> Digestion of pMTL007 using the whole cell extracts of the wild type strain DSM1731 and the mutant SMB009 respectively. <b>B.</b> Digestion of pMTL007 using the protoplast extracts of the wild type strain DSM1731 and the mutant SMB009.</p

Genetic manipulation of <i>C. acetobutylicum</i> SMB009 using unmethylated DNA.

<p><b>A.</b> Overexpression of <i>fdh</i> gene in <i>C. acetobutylicum</i> SMB009. The cells were grown to OD600 = 2∼3 in mRCM broth containing 50 µg/ml of erythromycin at 37°C. The boiled cell lysates were analyzed by SDS-PAGE (12% polyacrylamide gel). The overexpressed protein FDH (theoretical molecular weight 40 kDa predicted by DNAMAN Version 5) is indicated by the arrow on the right. <b>B.</b> Disruption of <i>adc</i> gene in <i>C. acetobutylicum</i> SMB009. Insertion of CTermB fragment into <i>adc</i> ORF was confirmed by PCR using the primers of 12-adc1 and 13-adc2 (corresponding to <i>adc</i> ORF positions 41-58 and 523-540 respectively). The insertion site of CTermB in <i>adc</i> ORF was validated by sequencing the PCR product of strain SMB009(<i>adc</i>::CTermB) amplified using the primers of 12-adc1 and 13-adc2.</p

Schematic representation of the construction of the pMTL009 for CAC1502 disruption.

<p>Schematic representation of the construction of the pMTL009 for CAC1502 disruption.</p

Engineering <i>in Vivo</i> Production of α‑Branched Polyesters

Polymers are an important class of materials that are used for a broad range of applications, from drug delivery to packaging. Given their widespread use, a major challenge in this area is the development of technology for their production from renewable sources and efforts to promote their efficient recycling and biodegradation. In this regard, the synthesis of polyesters based on the natural polyhydroxy­alkanoate (PHA) pathway offers an attractive route for producing sustainable polymers. However, monomer diversity in naturally occurring polyesters can be limited with respect to the design of polymers with material properties suitable for various applications. In this work, we have engineered a pathway to produce α-methyl-branched PHA. In the course of this work, we have also identified a PHA polymerase (CapPhaEC) from activated sludge from wastewater treatment that demonstrates a higher capacity for incorporation of α-branched monomer units than those previously identified or engineered. Production in Escherichia coli allows the construction of microbial strains that produce the copolyesters with 21–36% branched monomers using glucose and propionate as carbon sources. These polymers have typical weight-average molar masses (Mw) in the range (1.7–2.0) × 105 g mol–1 and display no observable melting transition, only relatively low glass transition temperatures from −13 to −20 °C. The lack of a melting transition indicates that these polymers are amorphous materials with no crystallinity, which is in contrast to the natural poly­(hydroxybutyrate) homopolymer. Our results expand the utility of PHA-based pathways and provide biosynthetic access to α-branched polyesters to enrich the properties of bio-based sustainable polymers

Primers used in this study.

<p>Primers used in this study.</p

Electrotransformation performance of <i>C. acetobuytlicum</i> DSM1731 and SMB009 with methylated pIMP1 (pIMP1(m)) or unmethylated pIMP1.

<p><b>A.</b> Strain DSM1731 transformed with pIMP1. <b>B.</b> Strain DSM1731 transformed with pIMP1(m). <b>C.</b> Strain SMB009 transformed with pIMP1. <b>D.</b> Strain SMB009 transformed with pIMP1(m). The italic numbers indicate corresponding transformation efficiency (10⁵ transformants/µg DNA) with standard deviations (n = 3) shown in parentheses. pIMP(m), methylated pIMP1 which was a mixture with pAN1.</p

Adjusting Surface Oxidized Layer of CoTe on PCN via In Situ N‑Doping Strategy to Promote Charge Separation of Z‑Scheme Heterojunction for Propelling Photocatalytic CO₂ Reduction

It has been a challenging issue to profoundly actuate the transfer and separation of photoinduced charge carriers by controlling the interface structure inside the heterojunction, owing to the molecular/subnanometric level interface region. Herein, a unique one-dimensional/two-dimensional (1D/2D) CoTe/PCN Z-scheme heterojunction is fabricated through the self-assembly of CoTe nanorods on the surface of polymeric carbon nitride (PCN) nanosheets. Significantly, in situ N-doping in the molecular/subnanometric surface oxidized layer of CoTe nanorods is achieved, effectively adjusting its chemical structure and element chemical states. Moreover, this N-doped surface oxidized layer can serve as a recombination region of photogenerated electrons from PCN and photogenerated holes from CoTe to increase the overall carrier separation efficiency in the Z-scheme heterojunction actuated by the built-in electric field. As a result, the photocatalytic CO2 reduction (CO2R) performance is enhanced dramatically, in which the yield of CO generated over the optimal 1D/2D CoTe/PCN heterojunction reaches up to triple than that over PCN. This unique contribution provides an emblematic paradigm for adjusting the interfacial structure of heterojunction and has a profound insight into the interfacial adjusting mechanism to improve the charge separation efficiency in the photocatalytic reaction