CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.

Co-biosynthesis of germacrene A, a precursor of ß-elemene, and lycopene in engineered Escherichia coli.

ß-Elemene is the major component of a traditional Chinese medicine (Rhizoma Curcumae) for cancer treatment, and plant extraction is the major methods currently. Biosynthesis of ß-elemene is a promising and attractive route due to its advantages, including environmentally friendly processes, renewable resources, and sustainable development. In this research, biosynthesis of germacrene A, direct precursor of ß-elemene, in Escherichia coli was successfully performed and 11.99 mg/L germacrene A was obtained. Thereafter, a cobiosynthesis system for germacrene A and lycopene, another kind of isoprenoid, was constructed. Furthermore, the cultivation conditions were optimized. The germacrene A production was increased to the highest level reported to date, 364.26 mg/L, threefold increase to the strain with only germacrene A production. The cobiosynthesis system was suggested to promote the metabolic flux for germacrene A production. This research enabled germacrene A production in E. coli, and it highlights the promoting mechanism of the cobiosynthesis system for two chemicals which are both belonging to isoprenoids. KEY POINTS : • Co-production of germacrene A and lycopene in E. coli. • Promoting mechanism of cobiosynthesis of two isoprenoid compounds in E. coli. • Shake-flask production of germacrene A reached to the highest 364.26 mg/L in E. coli.

Hydrophobic cell surface display system of PETase as a sustainable biocatalyst for PET degradation.

Remarkably, a hydrolase from Ideonella sakaiensis 201-F6, termed PETase, exhibits great potential in polyethylene terephthalate (PET) waste management due to it can efficiently degrade PET under moderate conditions. However, its low yield and poor accessibility to bulky substrates hamper its further industrial application. Herein a multigene fusion strategy is introduced for constructing a hydrophobic cell surface display (HCSD) system in Escherichia coli as a robust, recyclable, and sustainable whole-cell catalyst. The truncated outer membrane hybrid protein FadL exposed the PETase and hydrophobic protein HFBII on the surface of E. coli with efficient PET accessibility and degradation performance. E. coli containing the HCSD system changed the surface tension of the bacterial solution, resulting in a smaller contact angle (83.9 ± 2° vs. 58.5 ± 1°) of the system on the PET surface, thus giving a better opportunity for PETase to interact with PET. Furthermore, pretreatment of PET with HCSD showed rougher surfaces with greater hydrophilicity (water contact angle of 68.4 ± 1° vs. 106.1 ± 2°) than the non-pretreated ones. Moreover, the HCSD system showed excellent sustainable degradation performance for PET bottles with a higher degradation rate than free PETase. The HCSD degradation system also had excellent stability, maintaining 73% of its initial activity after 7 days of incubation at 40°C and retaining 70% activity after seven cycles. This study indicates that the HCSD system could be used as a novel catalyst for efficiently accelerating PET biodegradation.

Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering.

Farnesene, as an important sesquiterpene isoprenoid polymer of acetyl-CoA, is a renewable feedstock for diesel fuel, polymers, and cosmetics. It has been widely applied in agriculture, medicine, energy, and other fields. In recent years, farnesene biosynthesis is considered a green and economical approach because of its mild reaction conditions, low environmental pollution, and sustainability. Metabolic engineering has been widely applied to construct cell factories for farnesene biosynthesis. In this paper, the research progress, common problems, and strategies of farnesene biosynthesis are reviewed. They are mainly described from the perspectives of the current status of farnesene biosynthesis in different host cells, optimization of the metabolic pathway for farnesene biosynthesis, and key enzymes for farnesene biosynthesis. Furthermore, the challenges and prospects for future farnesene biosynthesis are discussed.

A Disjointed Pathway for Malonate Degradation by Rhodopseudomonas palustris.

The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl-CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure.IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently.